Cellular Targeted Label Delivery System

ABSTRACT

The present invention relates to an isolated cellular targeted delivery system comprising a CD45+ leukocyte cell comprising within said cell a complex of one or more iron binding proteins and/or a label as well as methods for producing such isolated cellular targeted delivery system and uses of such system for therapy diagnosis and in particular for diagnosis of cancer, particularly metastatic cancer, in particular for therapy of cancer.

The present invention relates to an isolated cellular targeted deliverysystem comprising a CD45⁺ leukocyte cell comprising within said cell acomplex of one or more iron binding proteins and/or a label as well asmethods for producing such isolated cellular targeted delivery systemand uses of such system for diagnosis and in particular for diagnosis ofcancer, particularly metastatic cancer.

BACKGROUND OF THE INVENTION

Current imaging tools are capable of detecting large metastases (largerthan 0.5-1 cm in size). However, they rarely detect the early spread ofmetastatic tumour cells. Human metastases smaller than 0.5 cm areavascular so without proper blood and oxygen supply. It means that thedelivery of contrast agents through the blood circulation for thepurpose of labelling these metastases and imaging them is not possible.The presence of hypoxia is a common characteristic of micrometastaseswhere hypoxic fraction may be as high as 90% with little or no bloodperfusion (Li, et al. 2012, Journal of Solid Tumours, 2(2): 28-33).Thus, severe hypoxia is considered as a general feature ofmicro-metastases.

The targeting of one or more micrometastases hidden within a largepopulation of normal cells presents a unique challenge since access tothe micrometastases is impeded by several bio-barriers, poor bloodsupply, further obstacles are presented by small size of themicrometastases and their dispersion to organs.

For the same reason micrometastases are often refractive to therapy.While the solid tumours from which the micrometastases have originatedoften respond well to conventional therapy there is often regrowth atthe site of the primary tumour or at sites of metastasis. Thisconstitutes a serious problem in clinical oncology (Muthana, et al.2012, Cancer Res; 73(2); 490-495). It is related to characteristics ofthe microenvironment of solid tumours that limit drug penetration,thereby exposing the tumour to lower than efficacious concentrations ofdrugs (Hobbs, et al. 1998, Proc Natl Acad Sci USA: 4607-4612). This iscaused by inadequate vasculature resulting in: high heterogeneity ofcancer cells, low oxygen tension (hypoxia), low pH and low glucoseconcentration within the mass (Kizaka-Kondoh, et al., 2003, Cancer Sci94(12):1021-1028). Additionally, rapid tumour cell proliferation in someareas might outpace the rate of new blood vessel growth, promotingformation of hypoxic area (Lewis and Murdoch, 2005, Am J Pathol167(3):627-635). This abnormal vessel architecture and, subsequently,their impaired function resulting in tumour hypoxia is associated with amore malignant phenotype and poor survival in patients suffering fromsolid tumours and results in both treatment failure due to decreaseddrug uptake and hypoxia-inducible changes in cancer cells (Sun, et al.,2012, Clin Cancer Res 18(3):758-770; Sullivan, et al., 2008 Mol CancerTher 7(7):1961-1973; Kizaka-Kondoh, et al., 2003, Cancer Sci94(12):1021-1028). Moreover, chemotherapy or radiotherapy causesadditional formation of large areas of tumour hypoxia thus making thetreatment of tumour even more difficult. The fact that the efficacy ofanticancer therapy is limited by the presence of hypoxic tumour cellshas resulted in the introduction of variety of therapeutic approachesaimed at overcoming this problem.

The present inventors have discovered that CD45⁺ leukocyte cells, inparticular activated macrophages, can uptake one or more labelscomplexed or not with one or more iron binding proteins in vitro anddeliver these complexes to or into cells, preferably to or into tumourcells in vivo. Based on this observation the present inventors haveovercome one or more of the above stated problems of the prior art.Thus, the targeted delivery system of the present invention providesinta alia one or more of the following advantages: (i) specific deliveryof one or more labels to tissues that attract above mentioned CD45⁺leukocytes, preferably into diseased cells, (ii) protection of labelsfrom inactivation in the blood circulation or clearance from the body,(iii) delivery of labels to, preferably into cells of poorly ornon-vascularized areas of disease, e.g. metastases, hypoxic areas withinlarger tumours, rheumatic lesions, avascular wounds, skin, (iv) reducedtoxicity of labels, (v) delivery of labels with poor pharmacokinetics,(vi) reduced side effects of the labels due to their targeted delivery,(vii) higher diagnostic efficacy with lower doses of the labels due totargeted delivery; (viii) lower risk of local tissue injury at the siteof label administration due to administration of the label linked withiron-binding protein, which is loaded inside the CD45⁺ leukocyte; and/or(ix) possibility to detect highly hypoxic small metastases(Pérez-Herrero E, Fernández-Medarde A. 2015, Eur J Pharm Biopharm93:52-79).

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to an isolated targeteddelivery system comprising a CD45⁺ leukocyte cell, which is preferablycapable of internalizing an iron binding protein comprising within saidcell a complex of one or more iron binding proteins and one or morelabels.

In a second aspect the present invention relates to an isolated targeteddelivery label system comprising a CD45⁺ leukocyte cell, preferablycapable of being labelled, comprising one or more labels, preferablyradiolabels or their conjugates and combinations.

In a third aspect the present invention relates to a method ofpreparation of the isolated targeted delivery system of the first aspectof the invention comprising steps of:

-   -   a) providing, preferably purified, iron binding protein;    -   b) covalently or non-covalently linking a label to and/or        encapsulating a label in an iron binding protein;    -   c) providing a CD45⁺ leukocyte cell; and    -   d1) incubating the CD45⁺ leukocyte cell in the presence of the        complex of the iron binding protein and the label produced in        step b) until the CD45⁺ leukocyte cell is at least partially        loaded with the complex of the iron binding protein and the        label produced in step b) and/or    -   d2) incubating CD45⁺ leukocyte cell in the presence of the label        until the CD45⁺ leukocyte cell is at least partially labelled        with the label.

In a fourth aspect the present invention relates to an isolated targeteddelivery system of the first aspect of the invention or producibleaccording to the method of the third aspect of the invention for use asa diagnostic.

In a fifth aspect the present invention relates to a diagnosticcomposition comprising the isolated targeted delivery system of thefirst aspect of the present invention or producible according to themethod of the third aspect of the invention and a pharmaceuticallyacceptable carrier and/or suitable excipient(s).

In a sixth aspect the present invention relates to the isolated targeteddelivery system of the first aspect of the present invention orproducible according to the method of the third aspect of the inventionfor use in diagnosing tumours, preferably a solid tumour and/or itsmetastases, preferably breast cancer, pancreatic cancer, bladder cancer,lung cancer, colon cancer or its metastases, or a tumour having hypoxicareas, inflammatory diseases or ischemic areas, in particular in skinwounds or after organ infarctus (heart) or ischemic retina.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

DEFINITIONS

To practice the present invention, unless otherwise indicated,conventional methods of chemistry, biochemistry, and recombinant DNAtechniques are employed which are explained in the literature in thefield (cf., e.g., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps. Asused in this specification and the appended claims, the singular forms“a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

The term “targeted label delivery” “targeted contrast agent delivery”refers to the delivery of a label, in particular a contrast agent to apatient or a person to be diagnosed which results in an increasedconcentration of the label, in particular the contrast agent in aparticular region of the body when compared to other regions of the bodyof that patient. Preferably, the relative concentrations are comparedbetween the diseased region(s) of the body and other regions of the bodyhaving similar access to the blood circulation. In preferred embodimentsthe concentration of the label, in particular a contrast agent in agiven number of cells or a given biopsy volume from the diseased regionis at least 10% higher, if compared to the identical number of cells orbiopsy volume from a non-diseased region after administration of thetargeted delivery system of the present invention, preferably after 1-24hrs. More preferably, the concentration of the label, in particular thecontrast agent in the diseased region of the body of a patient is atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70% at least 80%, at least 90%, at least 100%, at least 150%, atleast 200%, at least 250%, at least 300%, at least 350%, at least 400%,at least 450%, at least 500%, more preferably at least 1000% higher thanin a non-diseased region of the body after administration of thetargeted delivery system of the present invention, preferably after 2-24hrs. When assessed on the basis of total body distribution it ispreferred that at least 5% of the label, in particular the contrastagent administered to a patient or a person to be diagnosed is deliveredto the diseased region of the body, preferably at least 10%, morepreferably at least 15%. The targeted delivery of the label, inparticular the contrast agent limits the potential deleterious effectsof an label, in particular the contrast agent to the diseased region ofthe body.

The terms “targeted label delivery system” or “targeted delivery system”are used synonymously in the present application and refer to a systemthat is capable of delivering a label, in particular a contrast agent tothe targeted region, i.e. of capable of targeted delivery, preferablywithin the body of a patient.

The term “label” as used in the context of the present invention refersto any kind of compound being suitable for diagnostic purposes.Preferred compounds are selected from a fluorescent dye, a radioisotopeand a contrast agent. A contrast agent is a dye or other substance thathelps to show abnormal areas inside the body. In one embodiment the termlabel refers to a compound that comprises a chelating agent which formsa complex with divalent or trivalent metal cations. Preferredradioisotopes/fluorescence emitting isotopes are selected from the groupconsisting of alpha radiation emitting isotopes, gamma radiationemitting isotopes, Auger electron emitting isotopes, X-ray emittingisotopes, fluorescent isotopes, such as ⁶⁵Tb, fluorescence emittingisotopes, such as ¹⁸F, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ^(99m)Tc, ¹⁴⁰La, ¹⁷⁵Yb,¹⁵³Sm, ¹⁶⁶Ho, ⁸⁸Y, ⁸⁹Zr, ⁹⁰Y, ¹⁴⁹Pm, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi,⁷²As, ⁷²Se, ⁹⁷Ru, 109Pd, ¹⁰⁵Rh, ^(101m15)Rh, ¹¹⁹Sb, ¹²⁸Ba, 123I, ¹²⁴I,¹³¹I, ¹⁹⁷Hg, ²¹¹At, ¹⁶⁹Eu, ²⁰³Pb, ²¹²Pb, ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁹⁸Auand ¹⁹⁹Ag as well as conjugates and combinations of above with proteins,peptides, small molecular inhibitors, antibodies or other compounds,e.g. ¹⁸F fluorodeoxyglucose (¹⁸F-FDG) or ⁶⁴Cu-porfirin. Preferredfluorescent dyes are selected from the following classes of dyes:Xanthens (e.g. Fluorescein), Acridines (e.g. Acridine Yellow), Oxazines(e.g. Oxazine 1), Cynines (e.g. Cy7/Cy 3), Styryl dyes (e.g. Dye-28),Coumarines (e.g. Alexa Fluor 350), Porphines (e.g. Chlorophyll B),Metal-Ligand-Complexes (e.g. PtOEPK), Fluorescent proteins (e.g APC,R-Phycoerythrin), Nanocrystals (e.g QuantumDot 705), Perylenes (e.g.Lumogen Red F300) and Phtalocyanines (e.g. IRDYE™700DX) as well asconjugates and combinations of these classes of dyes or fluorescent ⁶⁵Tbemitting. Preferred contrast agents are selected from paramagneticagents, e.g. Gd, Eu, W and Mn, preferably complexed with a chelatingagent. Further options are superparamagnetic iron (Fe) complexes andparticles, compounds containing atoms of high atomic number, i.e. iodinefor computer tomography (CT), microbubbles and carriers such asliposomes that contain these contrast agents.

The term “peptide” or “polypeptide” is used interchangeably in thecontext of the present invention to refer to a chain of at least twoamino acids linked by peptide bonds. Thus, the term “peptide” in thecontext of the present invention is also used to refer to amino acidchains with more than 50, more than 100 or more than 150 amino acids.

The term “sequence identity” is used throughout the specification withregard to polypeptide and nucleotide sequence comparisons. In case wheretwo sequences are compared and the reference sequence is not specifiedin comparison to which the sequence identity percentage is to becalculated, the sequence identity is to be calculated with reference tothe longer of the two sequences to be compared, if not specificallyindicated otherwise. If the reference sequence is indicated, thesequence identity is determined on the basis of the full length of thereference sequence indicated by SEQ ID, if not specifically indicatedotherwise. For example, a polypeptide sequence consisting of 200 aminoacids compared to a reference 300 amino acid long polypeptide sequencemay exhibit a maximum percentage of sequence identity of 66.6% (200/300)while a sequence with a length of 150 amino acids may exhibit a maximumpercentage of sequence identity of 50% (150/300). If 15 out of those 150amino acids are different from the respective amino acids of the 300amino acid long reference sequence, the level of sequence identitydecreases to 45%. The similarity of nucleotide and amino acid sequences,i.e. the percentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, preferably with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) orwith the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. onhttp://www.ebi.ac.uk/Tools/clustalw/ or onhttp://www.ebi.ac.uk/Tools/clustalw2/index.html or onhttp://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html.Preferred parameters used are the default parameters as they are set onhap://www.ebi.ac.uk/Tools/clustalw/ orhttp://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). BLAST protein searches are performed with the BLASTPprogram, score=50, word length=3. To obtain gapped alignments forcomparative purposes, Gapped BLAST is utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programsare used. Sequence matching analysis may be supplemented by establishedhomology mapping techniques like Shuffle-LAGAN (Brudno M.,Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields.Structure based alignments for multiple protein sequences and/orstructures using information from sequence database searches, availablehomologs with 3D structures and user-defined constraints may also beused (Pei J, Grishin NV: PROMALS: towards accurate multiple sequencealignments of distantly related proteins. Bioinformatics 2007,23:802-808; 3DCoffee@igs: a web server for combining sequences andstructures into a multiple sequence alignment. Poirot O, Suhre K,Abergel C, O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul 1;32:W37-40.). When percentages of sequence identity are referred to inthe present application, these percentages are calculated in relation tothe full length of the longer sequence, if not specifically indicatedotherwise.

The term “leukocyte” is used in the context of the present invention torefer to cells of the immune system that are involved in protecting thebody against both infectious disease and foreign invaders. Allleukocytes are produced and derived from multipotent cells in the bonemarrow known as a hematopoietic stem cells. Leukocytes are foundthroughout the body, including the blood and lymphatic system. Allleukocytes have nuclei, which distinguishes them from the other bloodcells, the anucleated red blood cells (RBCs) and platelets. Types ofleukocyte can be classified in standard ways. Two pairs of the broadestcategories classify them either by structure (granulocytes oragranulocytes) or by cell division lineage (myeloid cells or lymphoidcells). These broadest categories can be further divided into the fivemain types: neutrophils, eosinophils, basophils, lymphocytes, andmonocytes. These types are distinguished by their physical andfunctional characteristics. Monocytes and neutrophils are phagocytic.Further subtypes can be classified; for example, among lymphocytes,there are B cells, T cells, and NK cells. Granulocytes are distinguishedfrom agranulocytes by their nucleus shape (lobed versus round, that is,polymorphonuclear versus mononuclear) and by their cytoplasm granules(present or absent, or more precisely, visible on light microscopy ornot thus visible). The other dichotomy is by lineage: Myeloid cells(neutrophils, monocytes, eosinophils and basophils) are distinguishedfrom lymphoid cells (lymphocytes) by hematopoietic lineage (cellulardifferentiation lineage).

The present inventors have observed that CD45⁺ expression ischaracteristic of leukocyte cells that are suitable to be used in thecontext of the targeted delivery system of the present invention, inparticular since CD45⁺ leukocyte cells are attracted to particulartissues and cells within the body and are capable of deliveringcomplexes of one or more iron binding proteins and one or more labels,in particular the contrast agents to or into cells. It is understood bythe skilled person that CD45⁺ leukocyte cells unless of clonal originare a mixed population of different leukocytes which share the commonproperty of expressing CD45⁺ surface antigen. Accordingly,subpopulations of cells within the diverse group of CD45⁺ leukocytes arecharacterized throughout the specification by further functional and/orstructural characteristics. The term “CD45⁺” indicates that the majorityof cells within a population of cells or essentially all cells expressthe CD45⁺ surface antigen. In this context and also with reference toother cellular surface antigens, the term “expresses” indicates that thesurface antigen is produced within the cell and detectably exposed onthe surface of a cell. The level of expression and, thus the number ofsurface antigens detectably exposed on the surface of a cell can varygreatly among different leukocytes. Generally, a cell is considered tobe positive, i.e. is indicated to be “⁺”, for a cellular surfaceantigen, if at least 5, preferably at least 10 copies of the surfaceantigen are detectably exposed on the surface of the cell. The skilledperson is well aware of how to detect, quantify and select for cells,which are positive (or negative) for a given cellular surface antigen.Preferred methods include Fluorescence Activated Cell Sorting (FACS). Inthis technology fluorescently labelled antibodies are used to bind tocellular surface antigens of a population of cells, the cells aresubsequently isolated into single cells and based on fluorescenceintensity measured for the single cell, characterized as being positiveor negative for the given cellular surface antigen. In some embodimentsof the present invention it is indicated that the expression of a givenprotein is high or low. This means that the protein is detectablyexpressed in both instances, i.e. is “⁺”, however, at different levels.High and low expression, respectively, will mean different absolutenumbers of proteins per cell for different proteins. Thus, a givenprotein may be considered to be expressed at high levels if there aremore than 500 detectable copies of that protein per cell and to beexpressed at low levels if there are between 1 to 50 detectable copiesof that protein per cell. However, another protein may be considered tobe expressed at high levels, if there are more than 5000 detectablecopies and expressed at low levels, if there are between 1 to 500detectable copies per cell. It is well known in the art how to quantifythe number of proteins expressed or produced in a cell using flowcytometry and Becton Dickinson Quantibrite™ bead method (see e.g. Pannu,K. K., 2001, Cytometry. 2001 Dec. 1; 45(4):250-8) or mass spectrometry(see, e.g. Milo, R., 2013, Bioessays, 35(12): 1050-1055). For thepurpose of the present invention the term “high expression” of a givenprotein refers to detectable expression of that protein that is at least70% of the highest expression level found, i.e. number of copies percell, in a population of healthy CD45⁺ leukocytes. The term “lowexpression” of a given protein refers to detectable expression of thatprotein that is 30% or less of the highest expression level found, i.e.number of copies of that protein per cell, in a population of healthyCD45⁺ leukocytes. Preferably, the “highest expression level” isdetermined as the average of the highest expression levels found inhealthy CD45⁺ leukocytes of different subjects. In some embodimentspreferred subpopulations of cells are characterized as “producing” agiven protein. This is understood to mean that the protein is notnecessarily detectable on the surface of the cell but may only bepresent inside the cell. The skilled person is well aware how to detectand/or quantify production of a protein inside a cell and/or selectcells producing such proteins.

The term “differentiated monocyte” is used in the context of the presentinvention to refer to a monocyte differentiated from the committedprecursor termed macrophage-DC precursor (MDP) mainly resident in bonemarrow (but could be also in the spleen) and differentiate into eitherdendritic cells or macrophages. In mice they consist of two mainsubpopulations: (i) CD11b⁺ cell with high expression of CX3CR1, lowexpression of CCR2 and Ly6C⁻ and (ii) CD11b⁺ cell with low expression ofCX3CR1, high expression of CCR2 and Ly6C⁺. After leaving the bonemarrow, mouse Ly6C⁺ monocytes differentiate into Ly6C⁻ monocytes incirculation. Similarly, in human monocyte differentiation, it isaccepted that CD14⁺⁺ classical monocytes leave bone marrow anddifferentiate into CD14⁺⁺CD16⁺ intermediate monocytes and sequentiallyto CD14⁺CD16⁺⁺non-classical monocytes in peripheral blood circulation(Yang et al. 2014; Biomark Res 2(1) doi. 10.1186/2050-7771-2-1).

Macrophages are tissue-resident professional phagocytes andantigen-presenting cells (APC), which differentiate from circulatingperipheral blood monocytes (PBMs). The term “activated macrophage” isused in the context of the present invention to refer to any macrophagethat is polarized. Macrophage activation is in general achieved byincubation with interleukins, cytokines and/or growth factors. Inparticular IL-4 and M-CSF can be used as activating agents. Activatedmacrophages of different phenotypes are classified into M1-macrophages,classically activated macrophages (CAM) and M2-macrophages,alternatively activated macrophages (AAM). The classically activatedM1-macrophages comprise immune effector cells with an acute inflammatoryphenotype. These are highly aggressive against bacteria and producelarge amounts of lymphokines (Murray, and Wynn, 2011, J Leukoc Biol,89(4):557-63). The alternatively activated, anti-inflammatoryM2-macrophages can be separated into at least three subgroups. Thesesubtypes have various different functions, including regulation ofimmunity, maintenance of tolerance and tissue repair/wound healing. Theterm “M1 inducer” is used in the context of the present invention torefer to a compound that directs differentiation of PBMs to macrophagesof the M1 type. The term “M2 inducer” is used in the context of thepresent invention to refer to a compound that directs differentiation ofPBMs to macrophages of the M2 type. The skilled person is aware of alarge number of ways to promote differentiation into either M1 or M2macrophages.

The term “phagocytosis by macrophages” is the process by which amacrophage engulfs a solid particle to form an internal vesicle known asa phagosome.

The term “iron binding protein” as used refers to a protein thatnon-covalently binds an iron ion. Examples of such proteins compriseferritin, haemoglobin, transferrin; and lactoferrin. Iron bindingproteins are bound by cellular surface receptors which facilitate theinternalization of these proteins into cells.

In a first aspect the present invention relates to an isolated targeteddelivery label system comprising a CD45⁺ leukocyte cell, preferablycapable of internalizing an iron binding protein, comprising within saidcell a complex of one or more iron binding proteins and one or morelabels, in particular the contrast agents.

It has been surprisingly found by the present inventors that CD45⁺leukocytes, preferably monocytes or activated monocytes and macrophages,preferably activated macrophages, preferably M1 macrophages, morepreferably M2 macrophages acquire label and deliver the label in theamount enough to be detected using imaging systems to allowing them tobe used to trace their location in the body. In case of ¹⁸F-FDG this wasparticularly preferable in case of monocytes and activated monocytes,macrophages, activated macrophages, preferably M1 macrophages and mostpreferably M2 macrophages. Administration of cells-loaded with a label(¹⁸F-FDG), preferably radioactive label can visualize organ withaccumulation of the labelled cells preferably by positron emissiontomography (PET) imaging. Accordingly, in a second aspect the presentinvention relates to an isolated targeted delivery label systemcomprising a CD45⁺ leukocyte cell, preferably capable of being labelledwith one or more labels, preferably radiolabels or their conjugates andcombinations. It is a preferred embodiment of the second aspect of thepresent invention that the cell is directly linked or labelled withlabel. The label maybe within said cell, or on its surface, preferablyit is on the surface of the cell.

The following preferred embodiments in each case further specify boththe first and second aspect of the present invention.

The ability of a given CD45⁺ leukocyte cell or cell population tointernalize iron binding proteins depends on the expression of receptorsinvolved in this internalization process. Receptors that lead tointernalization of ferritin comprise, e.g. TfR, CXCR4, CD163, and TIM-2.The skilled person is well aware how to measure the amount of uptake ofan iron binding protein and preferred method of measuring the uptake aredescribed in the Example Section below. The present inventors also notedthat subpopulations of CD45⁺ leukocyte cells have a certain propensityto internalize one iron binding protein over another iron bindingprotein and, thus can attain higher complex concentrations and/or showless leakage of the complex from the cells. Such CD45⁺ leukocytesubpopulations are described in more detail below.

The phrase “complex of one or more iron binding proteins and an label”as used in the context of the present invention refers to a compositionin which one or more molecules of the label are covalently ornon-covalently bound to one or more iron binding proteins. The covalentor non-covalent binding between the one or more iron binding proteinsand the one more label can be direct or indirect. In the latter case thelabel is linked to the iron binding protein via a linker or spacer.Linker or spacers are known to the skilled artisan, such as polyalanine,polyglycin, carbohydrates, (CH₂)n groups or polypeptide linkers. Theskilled artisan will, thus, be able to select the respective suitablelinker(s) or spacer(s) depending on the respective application. If theiron binding proteins form cages like, e.g. ferritin, than the term“complex” also encompasses the enclosure of label within the cage evenin the absence of a covalent or non-covalent bond between the protein(s)and the active compound(s). The formation of the complex allows thetransport of the label into the cell when the cell is internalizing theiron binding protein. Thus, it is preferred that the label are bound tothe iron binding protein in a way that does not interfere with thetransport mechanism. This can be easily tested by the skilled personusing uptake assays known in the art and described in the ExampleSection below. It is preferred that the complex is sufficiently stableto survive the transport within the cell to the target region within thebody. Thus, it is preferred that the complex rather than the label aloneis delivered to the cells or into the cells in the target region. Thisproperty also reduces possible deleterious effects, e.g. cytotoxicity,of the label to the CD45⁺ leukocyte. If label are covalently coupled tothe iron binding proteins such coupling is preferably through aminoacids residues known to be located in surface areas that are notinvolved in binding to the cellular receptors required for cellularuptake of the iron binding proteins. Iron binding proteins used in thecontext of the present invention can form stable non-covalently boundcomplexes with a wide variety of labels.

The CD45⁺ leukocyte originate from the patient to be treated in suchcase the cell loaded with the complex would be autologous to thepatient. It is also envisioned that patients are MHC typed prior totreatment with the targeted delivery of the present invention and thatthe leukocyte cell type used for a given patient is MHC matched to thepatient. In these two preferred embodiments the CD45⁺ leukocyte is aprimary cell or derived by a low number of differentiation steps from aprimary cell. Alternatively, the CD45⁺ leukocyte may be from animmortalized but preferably non-transformed CD45⁺ leukocyte cell line.Thus the blood used for CD45⁺ leukocyte, preferably macrophage isolationis preferably obtained from the patient to be treated or healthy donor.Alternatively the blood can be obtained from the blood bank. Use ofumbilical cord blood is also considered herein.

The present inventors noted that a subpopulation of CD45⁺ leukocytes,which are producible from a CD34⁺ hematopoietic precursor cell areparticular suitable for target specific delivery of the label.Accordingly, it is preferred that the leukocytes used to produce thetarget delivery system are derived from CD34⁺ hematopoietic precursorcells. The skilled person is well aware how to select CD34⁺hematopoietic precursor cells and how to differentiate such cells intoleukocytes.

Preferably, the CD45⁺ leukocyte is selected from the group consisting ofa monocyte, a differentiated monocyte, lymphocyte and a granulocyte.Preferred subpopulations in these general categories of leukocytes aredefined in the following by structural parameters, e.g. presence orabsence of a given protein, functional properties and/or method of theirproduction/differentiation. As outlined above, the targeted deliverysystem of the present invention still provides the advantages outlinedabove, if in a population of cells not every cell has a particularproperty in as long as the majority of cells within that population hasthat property. Thus, in the following the property of one preferred cellof the targeted delivery system of the present invention is described.It is appreciated by the skilled person that a pharmaceuticalcomposition of the present invention will comprises millions of cellsand that not every cell within the population will have the functionaland/or structural properties outlined herein but that the pharmaceuticalcomposition can nevertheless be used to treat a disease, if the majorityof cells share the respective functional and/or structural properties.

The present inventors have recognized that subpopulations of CD45⁺leukocytes have particular advantageous properties including amongothers efficiency and/amount of complex uptake in general, ability toretain the complex within the cell, i.e. to avoid leakage and of targetrelease of the label, efficiency of uptake of a particular iron bindingprotein and/or targeting to particular tissues or cells and, thussuitability to treat or ameliorate a particular disease. The presentinventors have, e.g. observed that CD45⁺ leukocytes, which express oneor more of the following antigens: CD204, CD206, CD200R, CCR2 have apreference for ferritin uptake over the uptake of other iron bindingproteins. Thus, if the iron binding protein in the complex is ferritinit is preferred to select CD45⁺ leukocytes that express one or more ofthe following antigens: CD204, CD206, CD200R, CCR2. Accordingly, in apreferred embodiment of the present invention

-   -   (i) the monocyte is a CD11b⁺ monocyte, preferably selected from        the group consisting of a CD11b⁺ CD14⁺ monocyte, a CD11b⁺ CD16⁺        monocyte, a CD11b⁺ CD14⁺ CD16⁺ monocyte, a CD11b⁺ CD14⁺ MHCII⁺        monocyte, a CD11b⁺ CD14⁺ CD115⁺ monocyte, CD11b⁺ CD114⁺        monocyte, CD11b⁺ CD116⁺ monocyte, CD11b⁺ CCR1⁺ monocyte, CD11b⁺        CCR2⁺ monocyte, CD11b⁺ CX3CR⁺ monocyte, CD11b⁺ CXR4⁺ monocyte,        CD11b⁺ CXR6⁺ monocyte and a CD11b⁺ CD14⁺ CD33⁺ monocyte;    -   (ii) the differentiated monocyte is selected from the group        consisting of a macrophage, an activated macrophage, preferably        a CD11b⁺ macrophage, more preferably a CD11b⁺ CD16⁺ macrophage,        CD11b⁺ CD32⁺ macrophage, CD11b⁺ CD64⁺ macrophage, CD11b⁺ CD68⁺        macrophage, preferably a CD11b⁺ CD86⁺ M1 macrophage, preferably        producing inducible nitric oxide synthetase (iNOS) and/or        secreting interleukin 12 (IL-12) or preferably CD11b⁺ CCR2⁺ M2        macrophage, CD11b⁺ CD204⁺ M2 macrophage, CD11b⁺ CD206⁺ M2        macrophage, CD11b⁺ CD204⁺ CD206⁺ M2 macrophage, CD11b⁺ Major        Histocompatibility Complex II⁺ (MHCII⁺) (low or hi expression)        M2 macrophage, CD11b⁺ CD200R⁺ M2 macrophage, CD11b⁺ CD163⁺ M2        macrophage or activated macrophage producing and/or secreting        Arginase-1 and/or interleukin 10 (IL-10); or a dendritic cell,        preferably with expression of CD11b CD11c, CD11b CD80, CD11c        CD80, CD11c CD86, CD11c MHCII and CD11c CD123, preferably the        differentiated monocyte is not a foam cell expressing        Lectin-like oxidized low-density lipoprotein receptor-1 (Lox1⁺),        C-X-C chemokine receptor type 7 (CXCR7⁺) and Nuclear factor        (erythroid-derived 2)-like 2 (NRF2⁺). A foam cell is a type of        macrophage that localize to fatty deposits on blood vessel        walls, where they ingest low-density lipoproteins and become        loaded with lipids giving them a foamy appearance. These cells        secrete various substances involved in plaque growth and their        death promotes inflammation, thereby contributing to        cardiovascular disease;    -   (iii) monocyte or activated monocyte expressing of at least one        chemokine receptor, preferably selected from the group        consisting of CCR1, CCR2, CXR4, and CXR6, or at least one growth        factor receptor, preferably selected from the group consisting        of macrophage colony stimulating factor Receptor (CD115),        granulocyte colony stimulating factor Receptor (CD114), and        granulocyte-macrophage colony stimulating factor Receptor        (consisting of CD116 and CD131); monocytes of these        characteristics are particular suitable to treat inflammatory        conditions and cancer;    -   (iv) the lymphocyte is selected from the group consisting of a        CD3⁺ and CD4⁺ or CD8⁺ T lymphocyte, or a CD19⁺, CD20⁺, CD21⁺,        CD19⁺ CD20⁺, CD19⁺ CD21⁺, CD20³⁰ CD21⁺, or CD19⁺ CD20⁺ CD21⁺ B        lymphocyte; or    -   (v) the granulocyte is selected from the group consisting of a        neutrophil, preferably a CD66b⁺ neutrophil, an eosinophil and a        basophil, preferably a CD193⁺ eosinophil.

In a preferred embodiment of the targeted delivery system of the presentinvention the activated macrophage:

-   -   (i) is producible by in vitro incubation of a monocyte or        macrophage or their precursors with a factor capable of altering        expression markers on macrophages, preferably    -   (a) with at least one M1 inducer,    -   (b) with at least one M2 inducer,    -   (c) or with a factor capable of altering the macrophages ability        to secrete cytokines, preferably IL-10 and IL-12, chemokines        and/or to produce iNOS, arginase or other immunomodulating        enzymes; examples of such factors are: activated platelets,        IL-4, IL-10, IL-13, immune complex of an antigen and antibody,        IgG, heat activated gamma-globulin, glucocorticosteroid, tumour        growth factor-β (TGF-β), IL-1R, CC-chemokine ligand 2 (CCL-2),        IL-6, Macrophage colony-stimulating factor (M-CSF), peroxisome        proliferator-activated receptor γ (PPARγ) agonist, leukocyte        inhibitory factor (LIF), adenosine, helminth and fungal        infection, lipopolysaccharide (LPS), interferon γ (INF-γ),        granulocyte macrophage colony stimulating factor (GM-CSF) and        viral and bacterial infection; in this respect it was observed        that activation of a monocyte with a M1 inducer, particularly        LPS will cause cell to express iNOS, that activation of a        monocyte with a M1 inducer, particularly LPS will cause cell not        to express Arginase-1, that activation of a monocyte with a M2        inducer, particularly IL-4 will cause cell to express        Arginase-1, and that activation of a monocyte with a M2 inducer,        particularly IL-4 will cause cell not to express iNOS,    -   (ii) is characterized by expression of at least one of following        antigens: CD64, CD86, CD16, CD32, high expression of MHCII,        and/or production of iNOS and/or IL-12;    -   (iii) is producible by in vitro incubation of a monocyte or        macrophage with a factor capable of inducing the ability of the        macrophage to phagocytose, e.g. IL-18, opsonins (for example        complement-derived proteins such as iC3b, immunoglobulin G),        calcitonin gene-related peptide (CGRP), lipopolysaccharide        (LPS), interferon γ (INF-γ), granulocyte macrophage colony        stimulating factor (GM-CSF), viral infection and/or bacterial        infection;    -   (iv) is characterized by expression of at least one of following        antigens: CD204, CD206, CD200R; CCR2, transferrin receptor        (TfR), CXC-motive chemokine receptor 4 (CXCR4), CD163, and/or T        cell immunoglobulin-domain and mucin-domain 2 (TIM-2), and/or        show low expression of MHCII; activated macrophages having these        properties are particularly suitable for complexes comprising        ferritin as the iron binding protein;    -   (v) has the ability to phagocytose; and/or    -   (vi) is capable of cytokine secretion, preferably of IL-12, or        IL-10, or production of inducible nitric oxide synthetase (iNOS)        (or other pro-inflammatory compounds), arginase or other        immunosuppressive/anti-inflammatory compounds.

In a preferred embodiment of the targeted delivery system of the presentinvention the M1 inducer for differentiating macrophages into M1macrophages is selected from the group consisting of lipopolysaccharide(LPS), interferon γ (INF-γ), granulocyte macrophage colony stimulatingfactor (GM-CSF) and viral and bacterial infection and the M2 inducer fordifferentiating macrophages into M2 macrophages is selected from thegroup consisting of IL-4, IL-10, IL-13, immune complex of an antigen andantibody, IgG, heat activated gamma-globulin, glucocorticosteroid,tumour growth factor-β (TGF-β), IL-1R, CC-chemokine ligand 2 (CCL-2),IL-6, Macrophage colony-stimulating factor (M-CSF), peroxisomeproliferator-activated receptor γ (PPARγ) agonist, leukocyte inhibitoryfactor (LIF), adenosine, helminth and fungal infection.

It has been surprisingly found by the present inventors that bothcomplex loaded M1 macrophages and M2 macrophages are suitable fortargeted active agent delivery into hypoxic tissues, preferably a tumouror its metastasis. In general we observed that 3 to 5% of theadministered M1 macrophages localized at the tumour site while about 35%of the M2 macrophages showed tumour specific targeting. However, thisgeneral advantage of M2 macrophages were offset when using a complexcomprising haemoglobin and label, since significantly larger amounts ofthis complex could be loaded into M1 macrophages than into M2macrophages. Generally this specific tropisms makes M2 macrophages moresuitable for treating tumour and diseases characterized by hypoxictissue.

In a preferred embodiment of the targeted delivery system of the presentinvention the monocyte:

-   -   (i) is producible from a CD34⁺ hematopoietic precursor cell;    -   (ii) is producible by in vitro incubation of monocytes with at        least one inducer, preferably M1 or M2 inducer, more preferably        at least one M2 inducer;    -   (iii) is characterized by expression of at least one of the        following antigens: TfR, CD163, TIM-2, CD14, CD16, CD33, and/or        CD115;    -   (iv) is characterized by expression of at least one of the        following antigens: TfR, CD163, TIM-2, CXCR4, CD14, and/or CD16;        and/or    -   (v) has the ability to phagocytose.

In this embodiment of the targeted delivery system of the presentinvention the M1 inducer for differentiating monocytes is selected fromthe group consisting of LPS, INF-γ, GM-CSF or viral or bacterialinfection or the M2 inducer for differentiating monocytes is selectedfrom the group consisting of IL-4, IL-10, IL-13, immune complex of anantigen and antibody, IgG, heat activated gamma-globulins,Glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonist,Leukocyte inhibitory factor (LIF), cancer-conditioned medium, cancercells, adenosine and helminth or fungal infection.

It has been surprisingly found by the present inventors that monocytesare suitable for targeted active agent delivery into hypoxic tissues,preferably the tumour or its metastasis while monocytes treated with M2activators are more suitable for targeted active agent delivery intohypoxic tissues, preferably the tumour or its metastasis. This specifictropisms make monocytes treated with M2 activators more suitable totargeting tumour and hypoxic sites.

In a preferred embodiment of the targeted delivery system of the presentinvention the lymphocyte:

-   -   (i) is obtainable from blood, spleen, or bone marrow or is        producible from a CD34⁺ precursor cell as known to the skilled        person and also described in the, e.g. Lefort and Kim, 2010, J        Vis Exp 40: 2017; Tassone and Fidler, 2012, Methods in Molecular        Biology 882: 351-357; Kouro et al. 2005, Current Protocols in        Immunology, 66:F22F.1:22F.1.1-22F.1.9.;    -   (ii) is an immunologically competent lymphocyte;    -   (iii) expresses antigen specific T cell receptors; and/or    -   (iv) is characterized by expression of at least one of the        following antigens: (a) CD3 and CD4 or CD8 or (b): CD19, CD20,        CD21, CD19 CD20, CD19 CD21, CD20 CD21, or CD19 CD20 CD21        antigen, and is preferably capable of producing immunoglobulins.

In a preferred embodiment of the targeted delivery system of the presentinvention the granulocyte:

-   -   (i) is obtainable from blood, spleen or bone marrow or        producible from a CD34⁺ precursor cell as described, e.g. in        Kuhs et al. 2015, Curr Protoc Immunol 111:7.23-1-7.23.16;        Coquery et al. 2012, Cytometry A 81(9): 806-814; Swemydas and        Lionakis 2013, J Vis Exp 77: 50586.;    -   (ii) is characterized by expression of at least one of the        following CD66b and/or CD193;    -   (iii) is a polymorphonuclear leukocyte characterized by the        presence of granules in its cytoplasm; and/or    -   (iii) is characterized by expression of at least one of the        following: TfR, CD163, TIM-2, and/or CXCR4.

In a preferred embodiment of the targeted delivery system of the presentinvention the iron binding protein is selected from the group consistingof ferritin, preferably heavy (H) type ferritin, light (L) ferritinand/or mitochondrial ferritin; haemoglobin, preferably haemoglobin A,haemoglobin AS, haemoglobin SC, haemoglobin C, haemoglobin D,haemoglobin E, haemoglobin F, haemoglobin H; haemoglobin-haptoglobincomplex, haemopexin, transferrin; and lactoferrin. The terms ferritin;haemoglobin, preferably haemoglobin A, haemoglobin AS, haemoglobin SC,haemoglobin C, haemoglobin D, haemoglobin E, haemoglobin F, haemoglobinH; haemoglobin-haptoglobin complex, hemopexin, transferrin; andlactoferrin encompass structural variants of the naturally occurringproteins and, thus relates to proteins that have at least 70%,preferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least 95%more preferably at least 100% of the ability of the respective wild-typeprotein to bind iron ion(s). The iron binding proteins used in thecontext of the present invention are preferably of mammalian, morepreferably mouse, rat, dog, ape, in particular, chimpanzee, or human,most preferably of human origin. Consensus sequences of the preferrediron binding proteins used in the context of the present invention areshown in FIG. 1 below. Preferred structural variants are based on thesequences indicated in FIG. 1. The residues marked with X vary amongdifferent mammalian ferritins, transferrins, and haemoglobins. Thealteration of these residues is not crucial for the ability of theproteins to bind to iron ions. Accordingly, it is preferred that aminoacid mutations or deletions effect one or more of the residues markedwith an X.

Plasma proteins have always been privileged carriers for the delivery ofactive pharma ingredients in cancer therapy. Thus, albumin, the mostabundant plasma protein, is currently used in therapeutic protocols forthe delivery of taxane molecules and doxorubicin derivatives (Larsen M Tet al. 2016, Mol Cell Ther 27; 4:3).

Human transferrin and ferritin proteins have been considered aseffective carriers for the delivery of small molecules to specificallytarget cancer cells. To date, in spite of considerable efforts, nosuccessful transferrin or ferritin complexes have however reached theclinic (Luck A N et al. 2013, Adv Drug Deliv Rev 65(8):1012-9).

Ferritin has a cage architecture and capability of iron-binding whichcould be used to encapsulate labels inside its cavity. Ferritins are notabundant in plasma, but can be readily produced in high yield asrecombinant proteins in common protein expression vectors such asEscherichia coli cells. Ferritins H- or L-chains are encoded as smallprotein monomer (21 kDa and 19 KDa for H and L chains, respectively)capable of a 24-mer assembly into a cage-like structure, delimiting a 8nm diameter cavity. The present inventors noted in the context ofworking on the present invention that H-ferritin homopolymers, representa preferred protein construct in order to specifically deliverencapsulated drugs to CD45⁺ leukocyte cells expressing TfR. FurthermoreH-ferritin targets complexed labels to the cell nucleus (and thereforedirectly delivers DNA-binding proteins into the nucleus.

Purified transferrin can be efficiently conjugated to various moleculesthrough covalent linkers that are appropriately released inside thecells (Beyer U et al. 1998, J Med Chem 41(15):2701-2708). In case oftransferrin, only lysine groups on the protein surface are readyavailable for covalent attachment.

Haemoglobin has been considered in the past as a carrier, due to itsversatility in chemical conjugation with various molecules, itsabundance and relative stability in the blood (Somatogen, 1993,WO1993008842 A1). Nevertheless, the lack of receptor targetingproperties did not foster biomedical applications other than bloodsubstitutes or antisickling agent. As a matter of fact, Hb can only berecognized by CD163 (haptoglobin/haemoglobin receptor) epitopes on theleukocytes, especially monocyte-macrophage origin. The CD45⁺ leukocyte,in particular macrophage based protein delivery, described in thisapplication moved haemoglobin center stage as a target specific carrierof labels. Haemoglobin can be readily covalently linked to appropriatelabels, host hydrophobic label molecules within the heme binding pocket,by exchanging heme free fluorescent porphyrines or porphyrins coupled toradiolabel or even transport small molecules combined with label linkedto the heme iron. Hb can be easily modified by selective attachment ofthe appropriate label conjugate to the beta93 cysteine residue, the onlytitratable cystein on the protein surface. Maleimido functionalizedlabels, are examples of labels that can be readily and specificallyattached to the relevant cys beta93 residue. Alternatively, lysineresidues on the Hb surface (at least 10 titratable lysine residues perHb tetramer) may be easily amenable to label conjugation throughcleavable succinimide linkers. Haemoglobin also offers a uniquecapability of releasing non covalently bound heme group at acidic pHvalues. Apo-haemoglobin thus obtained is capable of hosting severalhydrophobic molecules within the empty heme pocket endowed with notablefluorescence properties (e.g. chlorine e6, hyperycin, phtalocyaninederivatives) (Dong J et al. 3 Photochem Photobiol B 2014, 140:163-172).

Whatever the conjugation/adsorption/binding method, haemoglobin (Hb),transferrin (Tf) and ferritins were shown by the present inventions tobe privileged carriers of labels, once loaded into appropriate cellsystems with tumour targeting properties, e.g. activated macrophages.The easy, fast, cheap and safe purification procedure of these proteinalso provide a tremendous added value.

Based on sequences of mammalian H-type ferritins, L-type ferritins,haemoglobin alpha chains, haemoglobin beta chains and transferrins aconsensus sequence was determined for each of these proteins. These areshown in FIG. 1 in SEQ ID NO: 2, 7, 9, 14, 16, 20, and 25,respectively). On this basis but also on the basis of deletion andstructural analysis disclosed in the prior art a minimal fragment wasdetermined for H-type ferritins, L-type ferritins, haemoglobin alphachains, and haemoglobin beta chains sufficient for uptake by CD45⁺leukocytes. These are shown in SEQ ID NO: 1, 3, 5 (H-type ferritin); 8,10, 12 (L-type ferritin), 15 and 17 (haemoglobin alpha chain) and 19 and21 (haemoglobin beta chain. Transferrin comprises a N-terminally locateddomain and a C-terminally located domain that are necessary for bindingiron and uptake by CD45⁺ leukocytes, if comprised in one polypeptide andpositioned between 100 to 450 amino acids apart, preferably between 150to 400, more preferably between 200 to 350 amino acids and morepreferably 250 to 320 amino acids apart. The N-terminal domain comprisesamino acids 1 to 82 of full length consensus transferrin (SEQ ID NO: 25)or full length human transferrin (SEQ ID NO: 28). The C-terminal domaincomprises amino acids 396 to 510 of full length consensus transferrin(SEQ ID NO: 25) or full length human transferrin (SEQ ID NO: 28). Ineach case an X is indicated in the consensus sequence it independentlystands for any amino acid and characterizes an amino acid not or onlypoorly conserved among mammalian H-type ferritins, which can be mutatedwithout or little detriment to the iron binding properties of therespective iron binding protein. It is preferred that X in each casetakes on the meaning of the amino acid of the respective human ironbinding protein aligning with X. This information can be taken, e.g.from FIG. 1, which shows alignments of the consensus sequences withhuman an in some instances mouse proteins.

Preferred H-type ferritins comprise or consist of the amino acidsequence indicated in SEQ ID NO: 1 and variants thereof having at least70% amino acid identity, more preferably at least 75% amino acididentity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a H-typeferritin consisting of the amino acid sequence according to SEQ ID NO: 1to be taken up by CD45⁺ leukocytes, preferably M2 leukocytes. Within SEQID NO: 1 X at position 5 may be present or absent, if present it meansany amino acid, preferably Ile, X at position 6 means any amino acid,preferably Asn, X at position 14 means any amino acid, preferably Tyr, Xat position 24 means any amino acid, preferably Tyr or Cys, X atposition 66 means any amino acid, preferably Phe, X at position 68 meansany amino acid, preferably Gin, X at position 75 means any amino acid,preferably Arg or Cys, X at position 90 means any amino acid, preferablyHis, X at position 94 means any amino acid, preferably Ser or Asn, X atposition 120 may be present or absent, if present it means any aminoacid, preferably His or Tyr, more preferably His, X at position 123means any amino acid, preferably Asn or Ser, more preferably Asn, X atposition 128 means any amino acid, preferably Ala or Ser, more prefrablyAla.

In a preferred embodiment the H-type ferritin comprises or consists ofmurine ferritin according to SEQ ID NO: 3. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a H-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 3 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the H-type ferritin comprises or consists ofhuman ferritin according to SEQ ID NO: 5. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a H-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 5 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the H-type ferritin comprises or consists of amammalian consensus sequence derived from aligning full length H-typeferritins according to SEQ ID NO: 2 or 7, with 2 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a H-type ferritin consisting of theamino acid sequence according to SEQ ID NO: 2 or 7, with 2 beingpreferred to be taken up by CD45⁺ leukocytes, preferably M2 macrophages.In SEQ ID NO: 2 X at position 6 can be any naturally occurring aminoacid, preferably Pro, X at position 14 can be any naturally occurringamino acid, preferably His, X at position 16 can be any naturallyoccurring amino acid, preferably Asp, X at position 21 may be present orabsent, if present it means any amino acid, preferably Ile, X atposition 22 means any amino acid, preferably Asn, X at position 30 canbe any naturally occurring amino acid, preferably Tyr, X at position 40can be any naturally occurring amino acid, preferably Tyr or Cys, morepreferably Tyr, X at position 82 can be any naturally occurring aminoacid, preferably Phe, X at position 84 can be any naturally occurringamino acid, preferably Gln, X at position 91 can be any naturallyoccurring amino acid, preferably Arg or Cys, more preferably Cys, X atposition 106 can be any naturally occurring amino acid, preferably His,X at position 110 can be any naturally occurring amino acid, preferablyAsn or Ser, more preferably Asn, X at position 137 can be any naturallyoccurring amino acid, preferably His or Tyr, more preferably His, X atposition 140 can be any naturally occurring amino acid, preferably Asnor Ser, more preferably Asn, X at position 145 can be any naturallyoccurring amino acid, preferably Ala or Ser, more preferably Ala, X atposition 164 can be any naturally occurring amino acid, preferably Alaor Ser, more preferably Ser, X at position 166 can be any naturallyoccurring amino acid, preferably Met or Leu, preferably Leu, X atposition 178 can be any naturally occurring amino acid, preferably Aspor His, more preferably Asp, X at position 181 is absent or anynaturally occurring amino acid, preferably Asn, X at position 182 isabsent or any naturally occurring amino acid, preferably Glu, X atposition 183 is absent or any naturally occurring amino acid, preferablySer. In SEQ ID NO: 7 X at position 6 can be any naturally occurringamino acid, preferably Pro X at position 14 can be any naturallyoccurring amino acid, preferably His, X at position 16 can be anynaturally occurring amino acid, preferably Asp, X at position 21 may bepresent or absent, if present it means any amino acid, preferably Ile, Xat position 29 can be any naturally occurring amino acid, preferablyTyr, X at position 81 can be any naturally occurring amino acid,preferably Phe, X at position 83 can be any naturally occurring aminoacid, preferably Gin, X at position 105 can be any naturally occurringamino acid, preferably His, X at position 144 can be any naturallyoccurring amino acid, preferably Ala or Ser, more preferably Ala, X atposition 180 is absent or any naturally occurring amino acid, preferablyAsn, X at position 181 is absent or any naturally occurring amino acid,preferably Glu, X at position 182 is absent or any naturally occurringamino acid, preferably Ser.

In a preferred embodiment the H-type ferritin comprises or consists ofmurine full length ferritin according to SEQ ID NO: 4 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a murine H-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 4 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the H-type ferritin comprises or consists ofhuman full length ferritin according to SEQ ID NO: 6 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human H-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 6 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

Preferred L-type ferritins comprise or consist of the amino acidsequence indicated in SEQ ID NO: 8 and variants thereof having at least70% amino acid identity, more preferably at least 75% amino acididentity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a L-typeferritin consisting of the amino acid sequence according to SEQ ID NO: 8to be taken up by CD45⁺ leukocytes, preferably M2 leukocytes. In SEQ IDNO: 8 X at position at position 5 can be any naturally occurring aminoacid, preferably Asp or Glu, more preferably Asp, X at position 12 canbe any naturally occurring amino acid, preferably Arg or Ser, morepreferably Ser, X at position 17 can be any naturally occurring aminoacid, preferably Ser or Arg, more preferably Ser, X at position 19 canbe any naturally occurring amino acid, preferably Arg or Gln, morepreferably Gln, X at position 29 can be any naturally occurring aminoacid, preferably Phe, X at position 30 can be any naturally occurringamino acid, preferably Tyr or Phe, more preferably Tyr, X at position 42can be any naturally occurring amino acid, preferably Ser or Gly, morepreferably Ser, X at position 56 can be any naturally occurring aminoacid, preferably Ala or Tyr, more preferably Tyr, X at position 61 canbe any naturally occurring amino acid, preferably Glu or Lys, morepreferably Lys, X at position 62 can be any naturally occurring aminoacid, preferably Met or Phe, more preferably Met, X at position 65 canbe any naturally occurring amino acid, preferably Asp or Gln, morepreferably Gln, X at position 75 can be any naturally occurring aminoacid, preferably Ile or Val, more preferably Ile, X at position 76 canbe any naturally occurring amino acid, preferably Lys or Gln, morepreferably Lys, X at position 79 can be any naturally occurring aminoacid, preferably Ala or Ser, more preferably Ala, X at position 80 canbe any naturally occurring amino acid, preferably Glu or Gln, morepreferably Gln, X at position 87 can be any naturally occurring aminoacid, preferably Pro or Gln, more preferably Pro, X at position 88 canbe any naturally occurring amino acid, preferably Glu or Asp, morepreferably Asp, X at position 91 can be any naturally occurring aminoacid, preferably Glu or Lys, more preferably Lys, X at position 94 canbe any naturally occurring amino acid, preferably Met or Leu, morepreferably Leu, X at position 96 can be any naturally occurring aminoacid, preferably Met or Leu, more preferably Met, X at position 99 canbe any naturally occurring amino acid, preferably Lys or Asn, preferablyLys, X at position 115 can be any naturally occurring amino acid,preferably Thr or Ala, more preferably Thr, X at position 119 can be anynaturally occurring amino acid, preferably Leu, X at position 125 can beany naturally occurring amino acid, preferably Ser or Thr, morepreferably Thr, X at position 127 can be any naturally occurring aminoacid, preferably Tyr or Phe, more preferably Phe, X at position 130 canbe any naturally occurring amino acid, preferably Lys or Glu, morepreferably Glu, X at position 140 can be any naturally occurring aminoacid, preferably Asp or Asn, more preferably Asp, X at position 146 canbe any naturally occurring amino acid, preferably Arg or His, morepreferably His, and X at position 148 can be any naturally occurringamino acid, preferably Leu or Val, more preferably Leu.

In a preferred embodiment the L-type ferritin comprises or consists ofmurine L-type ferritin according to SEQ ID NO: 10. Accordingly,preferred structural variants have at least 70% amino acid identity,more preferably at least 75% amino acid identity, more preferably atleast 80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a L-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 10 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the L-type ferritin comprises or consists ofhuman ferritin according to SEQ ID NO: 12. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a L-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 12 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the L-type ferritin comprises or consists of amammalian consensus sequence derived from aligning full length H-typeferritins according to SEQ ID NO: 9 or 14, with 9 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a L-type ferritin consisting of theamino acid sequence according to SEQ ID NO: 9 or 14, with 9 beingpreferred to be taken up by CD45⁺ leukocytes, preferably M2 macrophages.In SEQ ID NO: 9 X at position 12 X at position 12 can be any naturallyoccurring amino acid, preferably Asp or Glu, more preferably Asp, X atposition 19 can be any naturally occurring amino acid, preferably Ser orArg, more preferably Ser, X at position 24 can be any naturallyoccurring amino acid, preferably Ser or Arg, more preferably Ser, X atposition 26 can be any naturally occurring amino acid, preferably Arg orGln, more preferably Gln, X at position 36 can be any naturallyoccurring amino acid, preferably Phe, X at position 37 can be anynaturally occurring amino acid, preferably Tyr or Phe, more preferablyTyr, X at position 47 can be any naturally occurring amino acid,preferably Ser or Gly, more preferably Ser, X at position 63 can be anynaturally occurring amino acid, preferably Ala or Tyr, more preferablyTyr, X at position 68 can be any naturally occurring amino acid,preferably Glu or Lys, more preferably Lys, X at position 69 can be anynaturally occurring amino acid, preferably Met or Phe, more preferablyMet, X at position 72 can be any naturally occurring amino acid,preferably Asp or Gln, more preferably Gln, X at position 82 can be anynaturally occurring amino acid, preferably Ile or Val, more preferablyIle, X at position 83 can be any naturally occurring amino acid,preferably Lys or Gln, more preferably Lys, X at position 86 can be anynaturally occurring amino acid, preferably Ala or Ser, more preferablyAla, X at position 87 can be any naturally occurring amino acid,preferably Glu or Gln, more preferably Gln, X at position 94 can be anynaturally occurring amino acid, preferably Pro or Gln, more preferablyPro, X at position 95 can be any naturally occurring amino acid,preferably Glu or Asp, more preferably Asp, X at position 98 can be anynaturally occurring amino acid, preferably Glu or Lys, more preferablyLys, X at position 101 can be any naturally occurring amino acid,preferably Met or Leu, more preferably Leu, X at position 103 can be anynaturally occurring amino acid, preferably Met or Leu, more preferablyMet, X at position 106 can be any naturally occurring amino acid,preferably Lys or Asn, preferably Lys, X can be any naturally occurringamino acid, X at position 126 can be any naturally occurring amino acid,preferably Leu, X at position 132 can be any naturally occurring aminoacid, preferably Ser or Thr, more preferably Thr, X at position 134 canbe any naturally occurring amino acid, preferably Tyr or Phe, morepreferably Phe, X at position 137 can be any naturally occurring aminoacid, preferably Lys or Glu, more preferably Glu, X at position 147 canbe any naturally occurring amino acid, preferably Asp or Asn, morepreferably Asp, X at position 153 can be any naturally occurring aminoacid, preferably Arg or His, more preferably His, X at position 155 canbe any naturally occurring amino acid, preferably Leu or Val, morepreferably Leu, X at position 161 can be absent or any naturallyoccurring amino acid, preferably Ala, X at position 163 can be absent orany naturally occurring amino acid, preferably Thr, X at position 166can be absent or any naturally occurring amino acid, preferably Pro, andX at position 168 can be any naturally occurring amino acid, preferablyGly or Ala, more preferably Ala. In SEQ ID NO: 14 X at position 36 canbe any naturally occuring amino acid, preferably Phe, X at position 37can be any naturally occurring amino acid, preferably Tyr or Phe, morepreferably Tyr, X at position 94 can be any naturally occurring aminoacid, preferably Pro or Gln, more preferably Pro, X at position 126 canbe any naturally occurring amino acid, preferably Leu, X at position 137can be any naturally occurring amino acid, preferably Lys or Glu, morepreferably Glu, X at position 147 can be any naturally occurring aminoacid, preferably Asp or Asn, more preferably Asp, X can be any naturallyoccurring amino acid, X at position 163 can be absent or any naturallyoccurring amino acid, preferably Thr, X at position 166 can be absent orany naturally occurring amino acid, preferably Pro, X at position 168can be any naturally occurring amino acid, preferably Gly or Ala, morepreferably Ala.

In a preferred embodiment the L-type ferritin comprises or consists ofmurine full length ferritin according to SEQ ID NO: 11 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a murine L-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 11 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the L-type ferritin comprises or consists ofhuman full length ferritin according to SEQ ID NO: 13 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human L-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 13 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal mammalian consensus sequence derived from aligning full lengthalpha haemoglobins according to SEQ ID NO: 15 Preferred comprise orconsist of the amino acid sequence indicated in SEQ ID NO: 15 andvariants thereof having at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of an alpha haemoglobin consisting of the amino acidsequence according to SEQ ID NO: 15 to be taken up by CD45⁺ leukocytes,preferably M1 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal human amino acid sequence derived of human alpha haemoglobinaccording to SEQ ID NO: 17. Accordingly, preferred structural variantshave at least 70% amino acid identity, more preferably at least 75%amino acid identity, more preferably at least 80% amino acid identity,more preferably at least 85% amino acid identity, more preferably atleast 90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of an alphahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 17 to be taken up by CD45⁺ leukocytes, preferably M1 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa mammalian consensus sequence derived from aligning full length alphahaemoglobins according to SEQ ID NO: 16. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of an alpha haemoglobins consisting of the amino acidsequence according to SEQ ID NO: 16 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofhuman full length alpha haemoglobin according to SEQ ID NO: 18.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human full length alphahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 18 to be taken up by CD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal mammalian consensus sequence derived from aligning full lengthbeta haemoglobins according to SEQ ID NO: 19 and variants thereof havingat least 70% amino acid identity, more preferably at least 75% aminoacid identity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a betahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 19 to be taken up by CD45⁺ leukocytes, preferably M1 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal human amino acid sequence derived of human beta haemoglobinaccording to SEQ ID NO: 21. Accordingly, preferred structural variantshave at least 70% amino acid identity, more preferably at least 75%amino acid identity, more preferably at least 80% amino acid identity,more preferably at least 85% amino acid identity, more preferably atleast 90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a betahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 21 to be taken up by CD45⁺ leukocytes, preferably M1 macrophages.

In a preferred embodiment the beta haemoglobin comprises or consists ofa mammalian consensus sequence derived from aligning full length betahaemoglobins according to SEQ ID NO: 20. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of an beta haemoglobins consisting of the amino acidsequence according to SEQ ID NO: 20 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the beta haemoglobin comprises or consists ofhuman full length beta haemoglobin according to SEQ ID NO: 22.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human full length beta haemoglobinconsisting of the amino acid sequence according to SEQ ID NO: 22 to betaken up by CD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the transferrin comprises or consists of amammalian consensus sequence derived from aligning full length alphahaemoglobins according to SEQ ID NO: 25. Thus, particularly, preferredtransferrins comprise or consist of the amino acid sequence indicated inSEQ ID NO: 25 and of variants thereof having at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a transferrin consisting of theamino acid sequence according to SEQ ID NO: 25 to be taken up by CD45⁺leukocytes preferably M1 macrophages.

In a preferred embodiment the transferrin comprises or consists of humantransferrin according to SEQ ID NO: 28. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a transferrin consisting of the amino acidsequence according to SEQ ID NO: 28 to be taken up by CD45⁺ leukocytes,preferably M1 macrophages.

The iron binding properties of transferrins are dependent on aN-terminally and C-terminally located domain. Thus, in a preferredembodiment the transferrin used in the present invention comprises atleast the N-terminal domain according to SEQ ID NO: 23 and theC-terminal domain according to SEQ ID NO: 24. Preferred transferrincomprise proteins that comprise the amino acid sequence indicated in SEQID NO: 23 and 24 as well as variants thereof having at least 70% aminoacid identity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a transferrin consisting of theamino acid sequence according to SEQ ID NO: 23 and 24 to be taken up byCD45⁺ leukocytes, preferably MI macrophages. SEQ ID NO: 23 or 24indicates a consensus sequence of mammalian transferrins.

Thus, in a preferred embodiment the transferrin used in the presentinvention comprises at least the N-terminal domain according to SEQ IDNO: 26 and the C-terminal domain according to SEQ ID NO: 27. Preferredtransferrin comprise proteins that comprise the amino acid sequenceindicated in SEQ ID NO: 26 and 27 as well as variants thereof having atleast 70% amino acid identity, more preferably at least 75% amino acididentity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a transferrinconsisting of the amino acid sequence according to SEQ ID NO: 26 and 27,respectively, to be taken up by CD45⁺ leukocytes, preferably M1macrophages.

Preferred ferritins, also comprise proteins that, irrespective of thegiven amino acid sequence, conform to the 24-mer subunit assembly of afour helix bundle protein module, falling within given sequencealignments of distantly related proteins as defined by 3D structurebased alignments.

It is a surprising observation of the present inventors that monocytes,macrophages and preferably M2 macrophages are able to uptake the amountof label enough to make them visible using imaging methods at the siteof their accumulation (e.g. in the tumour or its metastasis, hypoxiasite). Surprisingly, inventors found out that lymphocytes and M2macrophages are better in uptake of complexes comprising one or moreferritin and one or more label, that M1 macrophages are better in uptakeof complexes comprising one or more haemoglobin and one or more labeland that macrophages are better in uptake of complexes comprising one ormore transferrin and one or more label. Accordingly, based on the tissueand cellular tropism of CD45⁺ leukocytes: monocytes, M1 and M2macrophages, granulocytes and lymphocytes, described above complexescomprising one or more ferritin and one or more label are used to loadM2 macrophages, lymphocytes or monocytes, if the tropism of M2macrophages, lymphocytes or monocytes is desired and complexescomprising one or more haemoglobin and one or more label are used toload M1 macrophages, if the tropism of M1 macrophages is desired.

In a preferred embodiment of the targeted delivery system of the presentinvention the label is selected from a fluorescent dye, a fluorescenceemitting isotope, a radioisotope, a detectable polypeptide or nucleicacid encoding such a detectable polypeptide and a contrast agent.

In a preferred embodiment of the targeted delivery system of the presentinvention the label comprises a chelating agent which forms a complexwith divalent or trivalent metal cations.

In a preferred embodiment of the targeted delivery system of the presentinvention the chelating agent is selected from the group consisting of1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid (DOTA),ethylenediaminetetraacetic acid (EDTA),1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),triethylenetetramine (TETA), iminodiacetic acid,Diethylenetriamine-N,N,N′,N′,N″-pentaacetic acid (DTPA) and6-Hydrazinopyridine-3-carboxylic acid (HYNIC).

In a preferred embodiment of the targeted delivery system of the presentinvention the contrast agent comprises a paramagnetic agent, preferablyselected from Gd, Eu, W and Mn, or ferrihydride.

In a preferred embodiment of the targeted delivery system of the presentinvention the radioisotope/fluorescence emitting isotope is selectedfrom the group consisting of alpha radiation emitting isotopes, gammaradiation emitting isotopes, Auger electron emitting isotopes, X-rayemitting isotopes, fluorescence emitting isotopes, such as ¹⁸F, ⁵¹Cr,fluorescent ⁶⁵Tb, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ^(99m)Tc, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁵³Sm,¹⁶⁶Ho, ⁸⁸Y, ⁹⁰Y, ¹⁴⁹Pm, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi, ⁷²As, ⁷²Se,⁹⁷Ru, ¹⁰⁹Pd, ¹⁰⁵Rh, ^(101m15)Rh, ¹¹⁹Sb, ¹²⁸Ba, ¹²³I, ¹²⁴I, ¹³¹I, ¹⁹⁷Hg,²¹¹At, ¹⁶⁹Eu, ²⁰³Pb, ²¹²Pb, ⁶⁴Cu, ⁸⁹Zr, ⁶⁷Cu, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁹⁸Au and¹⁹⁹Ag as well as conjugates and combinations of above with proteins,peptides, small molecular inhibitors, antibodies or other compounds(e.g. ¹⁸F-FDG or ⁶⁴Cu-porfirin).

In a preferred embodiment of the targeted delivery system of the presentinvention the fluorescence dye is selected from the group consisting ofthe following classes of fluorescent dyes: Xanthens, Acridines,Oxazines, Cynines, Styryl dyes, Coumarines, Porphines,Metal-Ligand-Complexes, Fluorescent proteins, Nanocrystals, Perylenesand Phtalocyanines as well as conjugates and combinations of theseclasses of dyes.

In a preferred embodiment of the targeted delivery system of the presentinvention the detectable polypeptide is an autofluorescent protein,preferably green fluorescent protein or any structural variant thereofwith an altered adsorption and/or emission spectrum.

As has been outlined above, the targeted delivery system of the presentinvention has particular suitability to deliver labels to hypoxic areas.

In a preferred embodiment of the isolated targeted delivery system ofthe present invention the bond(s) between the iron binding protein(s)and the label comprised in the complex are covalent and/or non-covalent;and/or the label comprised in the complex is entrapped/encapsulated bythe iron binding protein, preferably ferritin or multimers thereof. Inone embodiment the covalent and/or non-covalent coupling is indirectthrough a linker or spacer. If the formation of covalent bonds isdesired, relevant thiol, amino or carboxyl groups of the iron bindingproteins are used to covalently couple labels directly or indirectly tothe one or more iron binding protein. It is also envisioned thatdifferent labels are comprised in the complex. For example, one type oflabel may be bound to an iron binding protein (non-covalently bound),while another type of label is encapsulated. This approach utilizesdifferent release rates of the labels from the iron binding protein oncedelivered to the targeted tissue and/or cells.

In a third aspect the present invention relates to a method ofpreparation of the isolated targeted delivery system of the presentinvention comprising the steps of

-   -   a) providing purified iron binding protein as defined above;    -   b) covalently or non-covalently linking a label to and/or        encapsulating a label in an iron binding protein;    -   c) providing a CD45+ leukocyte cell as defined above; and    -   d1) incubating the CD45+ leukocyte cell in the presence of the        complex of the iron binding protein and the label produced in        step b) until the CD45+ leukocyte cell is at least partially,        preferably fully loaded with the complex of the iron binding        protein and the label produced in step b); and/or    -   d2) incubating CD45⁺ leukocyte cell in the presence of the label        until the CD45⁺ leukocyte cell is at least partially labelled        with the label.

In a further aspect the present invention relates to the isolatedtargeted delivery system producible by the method according to thesecond aspect of the present invention.

The formation of the adduct between the protein and the label may be anon-covalent label binding to the target protein and can be described asfollows: In the case of ferritin, labels can be typically encapsulatedwithin the internal cavity (physical confinement) by exploiting theassociation dissociation properties of the ferritin macromoleculeitself. Label molecules are held in place by non-covalent interactionswith amino acid residues within the cavity internal surface. Haemoglobinmacromolecules also offer the possibility of non-covalent binding ofselected labels molecules that may be hosted within the heme bindingpocket of haemoglobin itself. The heme can be displaced by the pocketand be replaced by labels with appropriate hydrophobicity profile. In afurther aspect, all proteins considered in the present invention may becovalently attached to labels modified by specific active linkermoieties reactive towards thiol or amino groups of the protein itself.As such, ferritins or haemoglobin may be linked to cysteine thiolreactive labels bearing a peptide based cleavable linker (e.g. cathepsinsensitive valine-citrulline sequence and para-aminobenzylcarbamatespacer). The peptide-based linker binds the protein to the label in astable manner so the label is not easily released from the protein underphysiologic conditions and help prevent toxicity and/or leakage tohealthy cells and ensure specificity of the labeling. The protein labeladduct thus generated is capable of attaching to the selected receptortypes, i.e. CD163 for haemoglobin and TfR for ferritin or transferrin,respectively. Once bound the protein label adduct is internalised byendocytosis and thus selectively taken up by targeted cells. The vesiclecontaining the label is fused with lysosomes and lysosomal cysteineproteases, particularly cathepsin B start to break downvaline-citrulline linker and the label is no longer bound to theantibody and is released directly into the tumour environment.

Alternatively, a label bearing a linker that specifically bind to lysineresidues generating a covalent complex with ferritin, haemoglobin ortransferrin in a reaction is used.

The term “full load” is used in the context of the present invention torefer to the maximum amount of iron binding protein, preferablyferritin, complexed with a label that can be taken up by the CD45+leukocyte cell, preferably macrophage more preferably activatedmacrophage.

In a fourth aspect the present invention relates to the isolatedtargeted delivery system of the first aspect of the present invention orproducible according to the method of the second aspect of the inventionfor use as a diagnostic.

In a fifth aspect the present invention relates to a diagnosticcomposition comprising the isolated targeted delivery system of thefirst aspect of the present invention or producible according to themethod of the second aspect of the invention and a pharmaceuticallyacceptable carrier and/or suitable excipient(s). Since the isolatedtargeted delivery system comprises living cells, it is preferred thatcarriers and excipients are chosen in such to keep the cells alive.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier”, as used herein, refers to a pharmacologicallyinactive substance such as but not limited to a diluent, excipient,surfactants, stabilizers, physiological buffer solutions or vehicleswith which the label is administered. Such pharmaceutical carriers canbe liquid or solid. Liquid carrier include but are not limited tosterile liquids, such as saline solutions in water and oils, includingbut not limited to those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Saline solutions and aqueous dextrose and glycerol solutions canalso be employed as liquid carriers, particularly for injectablesolutions. A saline solution is a preferred carrier when thepharmaceutical composition is administered intravenously. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

Suitable pharmaceutical “excipients” include starch, glucose, lactose,sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like.

“Surfactants” include anionic, cationic, and non-ionic surfactants suchas but not limited to sodium deoxycholate, sodium dodecylsulfate, TritonX-100, and polysorbates such as polysorbate 20, polysorbate 40,polysorbate 60, polysorbate 65 and polysorbate 80.

“Stabilizers” include but are not limited to mannitol, sucrose,trehalose, albumin, as well as protease and/or nuclease antagonists.

“Physiological buffer solution” include but are not limited to sodiumchloride solution, demineralized water, as well as suitable organic orinorganic buffer solutions such as but not limited to phosphate buffer,citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPESbuffer ([4 (2 hydroxyethyl)piperazino]ethanesulphonic acid) or MOPSbuffer (3 morpholino-1 propanesulphonic acid). The choice of therespective buffer in general depends on the desired buffer molarity.Phosphate buffer are suitable, for example, for injection and infusionsolutions.

In a sixth aspect the present invention relates to the isolated targeteddelivery system of the first aspect of the present invention orproducible according to the method of the second aspect of the inventionfor use in diagnosing tumours, preferably a solid tumour and/or itsmetastases, preferably breast cancer, pancreatic cancer, bladder cancer,lung cancer, colon cancer or its metastases, or a tumour having hypoxicareas, inflammatory disease or ischemic areas, in particular in skinwounds, other wounds, or after organ infarctus (heart) or ischemicretina.

The targeted delivery system according to the present invention enablestumour delivery of the labels, preferably contrast agents, whichnormally would not be able to reach the tumour (for example, due tosolubility problems or lack of vasculature). It also enables thedelivery of labels, preferably contrast agents to the hypoxic tumours orto the hypoxic areas of the tumour. This system also provides fordelivery of a contrast agent to any area within an organism subjected tohypoxic conditions, for example during ischaemic incidents, orundergoing an inflammatory process.

As mentioned above the present invention provides also a method fortargeted label delivery into the tumour mass. This method comprisespreparation of CD45⁺ leukocytes which enables highly efficient labeluptake or iron-binding protein (ferritin, haemoglobin and/ortransferring) uptake by the macrophages, wherein said ferritin,haemoglobin and/or transferrin carry an label, preferably contrast agentand tumour targeting.

Additionally, the present invention allows for targeting radiolabelsinto the tumour mass. These methods comprise CD45+ leukocytes,preferably activated macrophages loaded with eitherferritin-encapsulated or decorated with the contrast agent for MRIimaging (e.g. ferryhydrite) or PET/SPECT imaging (e.g. α or β radiationemitting radioisotope, which also emit a cell damaging amount of γradiation, preferably selected from the group consisting oflutetium-177, ytterbium-90, iodine-131, samarium-153, phosphorus-32,caesium-131, palladium-103, radium-233, iodine-125, and boron-10 or acell damaging amount of α radiation, preferably selected from the groupconsisting of actinium-225, bismuth-213, lead-212, and polonium-212).

The present invention exploits CD45⁺ leukocytes, preferably activatedmacrophages loaded with iron-binding proteins linked with a label,preferably contrast agent as a delivery system to target the tumour.Alternatively, CD45⁺ leukocytes, preferably activated macrophageslabelled with the label, preferably contrast agent constitute thedelivery label system to target the tumour and be visualised usingimaging methods (e.g. PET). Difficulties in their detection usingimaging methods are mainly related to an altered penetration of thecontrast agents to the hypoxic areas observed mostly in solid tumours,especially their micrometastases due to poor vasculature. However, theseavascular regions attract CD45⁺ leukocytes, preferably activatedmacrophages to migrate even in areas far away from blood vessels.Therefore, they constitute a delivery system of particles to the tumourmass. A promising example of such particles is iron-binding protein.However, when used as single agents they do not reach hypoxic regions,similarly to other compounds and accumulate in other organs.

The present inventors linked labels, or isotopes to haemoglobin ortransferrin using chemical methods and loaded it into CD45⁺ leukocytes(monocytes, macrophages, lymphocytes and/or granulocytes), preferablyactivated macrophages treating cells with iron-binding protein solutionas it is described in examples. The inventors observed that uponadministration to the animal, loaded CD45⁺ leukocytes, preferablyactivated macrophages migrate to the tumour hypoxic sites and releaseiron-binding protein with encapsulated labels into the cancer cells.This method allows precise administration of the labels to the tumoursite (especially to the hypoxic regions), avoiding their accumulation inother organs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Panel (A) shows a minimal active fragment of a consensus aminoacid sequence among mammalian ferritin H chains and two full lengthconsensus sequences based on several mammalian ferritin H chains (seeSEQ ID NO: 1, 2 and 7, respectively) as well as a minimal and fulllength amino acid sequence of mouse (SEQ ID NO: 3 and 4) and human (SEQID NO: 5 and 6) ferritin H chain. Panel (B) shows a a minimal activefragment of a consensus amino acid sequence among mammalian ferritin Lchains and two full length consensus sequences based on severalmammalian ferritin L chains (see SEQ ID NO: 8, 9 and 14, respectively)as well as a minimal and full length amino acid sequence of mouse (SEQID NO: 10 and 11) and human (SEQ ID NO: 12 and 13) ferritin L chain.Panel (C) shows a minimal active fragment of a consensus amino acidsequence among mammalian haemoglobin alpha chains and one full lengthconsensus sequences based on several mammalian haemoglobin alpha chain(see SEQ ID NO: 15 and 16, respectively) as well as a minimal and fulllength amino acid sequence of human (SEQ ID NO: 17 and 18) haemoglobinalpha chain. Panel (D) shows a minimal active fragment of a consensusamino acid sequence among mammalian haemoglobin beta chains and a fulllength consensus sequences based on several mammalian haemoglobin betachain (see SEQ ID NO: 19 and 20, respectively) as well as a minimal andfull length amino acid sequence of human (SEQ ID NO: 21 and 22)haemoglobin beta chain. Panel (E) shows a N- and C-terminal minimalactive fragment of a consensus amino acid sequence among mammaliantransferrins (SEQ ID NO: 23 and 24) and a full length consensussequences based on several mammalian transferrins (SEQ ID NO: 25) aswell as a N- and C-terminal minimal active fragment of a humantransferrin (SEQ ID NO: 26 and 27) and full length amino acid sequenceof human transferrin (SEQ ID NO: 28). In the consensus sequences Xindicates a position that is variable and stands for any natural aminoacid. Preferably, in each case X in dependently of other X stands forthe amino acid present in the human protein.

FIG. 2: Shows macrophage inside the mouse tumour mass (TRITC stainedbefore injection, loaded with FITC-decorated ferritin).

FIG. 3: Shows confocal microscopy image of tumour tissue mouse injectedwith mammary cancer cells and given i.v. macrophages loaded withFITC-ferritin (asterix)—it is clearly observed, not only in macrophagesbut also in cancer cells, that ferritin-FITC spread within all tumourmass.

FIG. 4: Shows MRI images of mouse mammary tumour. The mouse was treated(at time point 0 h) with macrophages (i.v. injection) loaded withferritin Fh. Then we observed increased diameter of blood vessels(arrow) filled with injected macrophages (giving significant T2-signalreduction) and afterword macrophages spread to the tissue (spot-likepattern; arrows). These changes (in the same time points) were observedin all examined mice.

FIG. 5: Shows ferritin, haemoglobin and transferrin uptake bymacrophages, ferritin and haemoglobin uptake by monocytes and ferritinuptake by lymphocytes and granulocytes.

FIG. 6: Shows the stability of the ferritin storage by macrophages.

FIG. 7: Shows the picture from two-photon microscopy showing tumour froma mouse that received pre-labeled (before administration) macrophagescontaining Ferritin-FITC.

FIG. 8: Shows the whole body imaging of mice that intravenously receivedlabeled macrophages, showing their accumulation in the tumour site andtheir distribution in other organs.

FIG. 9: Shows the migration of macrophages to hypoxic tissue, across-section of the tumour from a mouse that was administeredintravenously with pre-labeled macrophages, tumour hypoxic areas arevisualized with a hypoxia marker—pimonidazolone.

FIG. 10: Shows the signal recorded by PET from a whole-body analysis ofmice with the metastatic 4T1 cancer. Mice received intravenouslymacrophages loaded with 18F-FDG. Signal accumulation is increased in thelungs of mice with micrometastases (confirmed by pathology examination).Mice receiving plain 18F-1-DG, or mice without 4T1 cancer had lower PETsignal.

EXAMPLE SECTION Example 1—Activation of Macrophages

Macrophages for use according to the present invention were obtained,differentiated and activated as follows. In order to activatemacrophages, they are obtained firstly from bone marrow precursors (forexample see paper: Weischenfeld and Porse, 2008, CSH Protoc, doi.10.1101/pdb.prot.5080) or blood monocytes. Alternatively, they can beobtained from peritoneum. The methods of macrophage isolation, culture,differentiation and polarization/activation are well known for thoseskilled in the art. For example, they have been described in details byMurray et al. (Immunity, 2014, 41(1):14-20).

In this practical realization of the invention bone marrow derivedmacrophages were obtained from BALB/c or C57B1/6 mouse, however canineblood-monocyte-derived macrophages or commercially available macrophagecell lines (monocyte-macrophage lineage mouse cells: RAW 264.7, J744,human cells: THP-1, U937, or canine cells DH82).

Shortly, such bone marrow derived macrophages are seeded in plasticPetri dish in 5 ml medium (3 ml cells per plate):DMEM:F12+glutamine/glutamax+10% FBS+Penicillin/Streptomycin and 20% ofL929 conditioned medium or M-CSF (50 ng/ml). In the next five days themedium is supplemented in growth factor and one of the activatingcompounds or their combinations as one cytokine cocktail.

Alternatively, macrophages have been cultured in “M1/M2 MacrophageGeneration Medium” (Promocell) or equivalent commercially available orself-made medium containing all the necessary cytokines and interleukinsto consider them as activated.

In order to obtain macrophages from blood monocytes, fresh blood (notolder than 12 hours) is spin down using Histopaque system 1077 orequivalent and white blood cells (or alternatively, only white bloodcells collected from the blood bank) in an appropriate amount ofpre-warmed Monocyte Attachment Medium (or equivalent, e.g. DMEM/RPMIsupplemented with M-CSF). e.g. 15 ml Medium per T-75 flask. A seedingdensity should be of 1-2 million/cm² for mononuclear cells with amonocyte content of ≥25% and 1.5-3 million/cm2 for a monocyte content of<25%. Then, cells are incubated for 1-1.5 hours at 5% CO₂ and 37° C. inthe incubator without any further manipulation.

After cell attachment, they are washed at least twice, and then anappropriate amount of complete “M1- or M2-Macrophage Generation MediumDXF” is added to the cells (e.g. 20 ml per T-75 flask) and cells areincubated for 6 days at 37° C. and 5% CO₂ without medium change. Inorder to activate macrophages, the whole medium should be replaced withmedium supplemented by activating compound.

Activating compounds used in this invention (for bone-marrow derivedcells or to activate cells from monocyte-macrophage cell lines) are asfollows: IL-4 (20 ng/ml), IFN-γ (at least 20 ng/ml), LPS (at least 10ng/ml), IL-13 (at least 20 ng/ml), IL-10 (at least 20 ng/ml),dexamethason (at least 20 μg/ml), oxLDL (at least 20 ng/ml), TNF-α (20ng/ml), TGF-β (20 ng/ml), cortisol (150-300 ng/ml) or their combinationsas one cytokine cocktail. In order to obtain unactivated macrophages,the activating compound has not been added.

Reverse of the polarization/activation of macrophages (from classicallyactivated to alternatively activated) can be reached for example byculture of macrophages in appropriate cytokines listed above for atleast 48 hrs.

Example 2—Monocyte Isolation

In order to obtain monocytes in this practical realization of theinvention bone marrow derived or spleen-derived monocytes were obtainedfrom BALB/c or C57B1/6 mouse, however canine blood monocyte orcommercially available monocyte cell lines were used(monocyte-macrophage lineage mouse cells: RAW 264.7, J744, human: THP-1,U937, or canine DH82 cell line).

To obtain blood monocytes, fresh blood (not older than 12 hours) is spindown using Histopaque system 1077 or equivalent and white blood cellsare seeded in an appropriate amount of pre-warmed Monocyte AttachmentMedium (or equivalent, e.g. DMEM/RPMI supplemented with 20 ng/ml M-CSF),e.g. 15 ml Medium per T-75 flask. Alternatively, only white blood cellscollected from the blood bank (buffy coat) may be used. A seedingdensity should be of 1-2 million/cm2 for mononuclear cells with amonocyte content of ≥25% and 1.5-3 million/cm2 for a monocyte content of<25%. Then, cells are incubated for 1-1.5 hours at 5% CO₂ and 37° C. inthe incubator without any further manipulation. After cell attachment,they are washed at least twice, and adherent cells are considered asmonocytes.

In order to obtain bone-marrow derived monocytes, in this practicalrealization of the invention bone marrow derived macrophages wereobtained from BALB/c or C57B1/6 mouse. Shortly, such bone marrow derivedprecursors are seeded in plastic Petri dish in 5 ml medium (3 ml cellsper plate): DMEM:F12+glutamine/glutamax+10% FBS+Penicillin/Streptomycinand 20% of L929 conditioned medium or 20 ng/ml M-CSF. Two days later 5ml of standard medium is added. Then, after two days 0.5 ml/plate L929conditioned medium is added. Adherent cells are considered as monocytes.

In order to obtain spleen derived monocytes, in this practicalrealization of the invention, the spleen has been mechanicallydissociated to obtain single cell suspension and passed through the 70μm cell strainer. Cells were centrifuged and supernatant was removed.After erythrocyte lysis the monocytes were isolated using magnetic beadpurification e.g. EasySep Mouse Monocyte Enrichment Kit protocol andappropriate magnet.

To obtain better effects of their protein loads and migration before usethey may be pre-treated with macrophage activation stimuli: IL-4 (20ng/ml), IFN-γ (at least 20 ng/ml), LPS (at least 10 ng/ml), IL-13 (atleast 20 ng/ml), IL-10 (at least 20 ng/ml), dexamethason (at least 20μg/ml), oxLDL (at least 20 ng/ml), TNF-α (20 ng/ml), TGF-β (20 ng/ml),cortisol (150-300 ng/ml) or their combinations as one cytokine cocktail.

Example 3—Granulocyte Isolation

To obtain granulocyte cells from blood, 9 parts of blood were dilutedwith 1 part of ACD buffer (containing 0.17 M d-glucose, 0.10 M citricacid, 0.11 M trisodium citrate). Blood from this step was furtherdiluted with PBS at the 1:1 ratio and centrifuged. After removing plasmaand buffy coat, remaining cells were mixed with PBS to 80% of theoriginal volume from the first step (ACD-blood) and then diluted withcold distillated water at the ratio of 4:12. Then, 6 parts of 2.7% ofNaCl solution were added and centrifuged. After removal of supernatantcells were resuspended in RPMI-1640 medium. These cell were consideredas granulocytes.

Example 4—Lymphocyte Isolation

In order to obtain spleen derived lymphocytes, in this practicalrealization of the invention, the spleen has been mechanicallydissociated to obtain single cell suspension and passed through the 70μm cell strainer. Cell were centrifuged and supernatant was removed.After erythrocyte lysis the lymphocytes were isolated using magneticbead purification e.g. EasySep Mouse CD4⁺ Enrichment Kit protocol andappropriate magnet.

Example 5—Preparation of Ferritin Complexes

In order to incorporate ferritins with the anticancer drug (e.g. classicdrugs like cyclophosphamide, chlorambucil, melphalan, bendamustine,banoxantrone or hypoxia-activated prodrug like TH-302) or with the“imaging contrast agents” (e.g. ferrihydride or isotope) ferritins haveto be prepared before macrophage treatment. Shortly, recombinant mouseproteins according to SEQ ID NO: 4 (FIG. 1) are obtained as follows. Theexpression vector pET-22b containing a synthetic gene encoding ferritinprotein of SEQ ID NO: 4 was transformed into E. coli BL21 (DE3). E. coliculture was grown at 37° C. to OD600 0.6 in 1 L of Luria-Bertani broth(LB) added with ampicillin (100 mg/L). Protein expression was induced byaddition of 1 mM isopropyl thio-b-D-galactoside (IPTG) and the culturewas incubated overnight. Cells were harvested by centrifugation (15000 gfor 15 min) and suspended in 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1mg/mL DNase, 10 mM MgCl₂ and disrupted by sonication. The lysate wascentrifuged at 15000 g for 30 mM and the supernatant was treated 10 minat 50° C., centrifuged to remove denatured proteins and then at 70° C.for 10 min and centrifuged again. The supernatant was added with 30%(NH)₄SO₄ at 4° C. stifling for 1 h and centrifuged at 15000 g for 30min. The supernatant was added with 70% (NH)₄SO₄ at 4° C. stirring for 1h and centrifuged at 15000 g for 30 mM. The pellet was resuspended in 20mM Hepes (pH 7.5), 150 mM NaCl and dialysed overnight at 4° C. againstthe same buffer. The protein was loaded on a HILOAD 26/600 SUPERDEX 200gel-filtration column (GE-Healthcare) and then sterile filtered andstored at 4° C. (FIG. 9) Protein concentration was determinedspectrophotometrically at 280 nm using a molar extinction coefficient of21000 M⁻¹ m⁻¹ and by Bradford assay measuring the absorbance at 595 nm.

Said ferritins include recombinant mammalian ferritin proteins H and/orL homopolymers.

Ferritins, obtained as previously described, are purified by standardmethods in order to obtain an endotoxin free, pre-clinical grade product(see, for example: Ceci et al. 2011, Extremophiles 15(3):431-439;Vanucci et al. 2012, Int J Nanomed 7:1489-1509). Shortly, the ferritinconserved sterile in a storage solution containing 20 mM Hepes pH 7.5 isdiluted to a final concentration of 4 uM in 24-mer in acidic solution(final pH<3.0) or, alternatively, at highly basic pH values (pH>9.5)(see for example Pontillo et al., 2016), thus allowing the dissociationof multimer. Drugs are dissolved at very high concentrations in theappropriate solvent and then a small volume is added to the ferritinsolution with a 200 molar excess. PH is then brought to neutrality byaddition of appropriate amounts of NaOH/HCl solutions in order to allowmultimer reconstitution. Current experimental methods indicate thatthree/four washings using PBS (concentration steps) in 100 kDa cut offconcentrators allows rapid and complete elimination both the co-solventsas well as non-encapsulated drugs and full recovery of drug loadedferritin nanocages. The ferritin-drug complex thus obtained was thenflash freezed in liquid nitrogen and lyophilised.

Depending on the choice of co-solvent and on the intrinsic chemicalproperties of the drug molecule, it can be estimated that up to 150-180drug molecules can be entrapped/adsorbed within the 24-mer ferritincage.

Labels may also be covalently coupled to ferritin amino acid side chains(lysines or cysteines) by appropriate choice of phenylhydrazone,succinimide or maleimide activated labesl. Accordingly, i)phenylhydrazone derivative may breaks and liberates the label from theferritin surface, ii) lysine bound derivatives may become active afterfull protein degradation into aminoacids or iii) cysteine boundderivative may be liberated within the cell through reductive hydrolysisof the maleimede thioether link.

Example 6—Preparation of Haemoglobin-Compound Complex

Human haemoglobin is prepared from fresh red cells as described inRossi-Fanelli et al. (Archives of biochemistry and biophysics77:478-492, 1958). Shortly, the heparinized blood, obtained from healthydonors, was centrifuged at 1600 rpm for 30 minutes (4° C.) to sedimentthe RBCs. Buffy coat was accurately removed by needle aspiration on thesurface of the pellet. The plasma supernatant was discarded and the RBCpellet was washed three times by resuspending the RBCs in isotonic 0.9%saline solution and centrifuging at 1600 rpm for 30 minutes at 4° C.After the final saline wash and centrifugation step, the RBC pellet wasresuspended in distilled water buffered at pH 7.2 with 5 mM potassiumphosphate buffer (PB, pH=7.2) and allowed to lyse at 4° C. overnightunder gentle stirring. Dialyzed RBC lysate was subsequently centrifugedat 13.000 rpm for 30 min at 4° C. and supernatant was directly loaded onan ÄKTA Explorer system equipped with an XK 26/40 column packed withQ-sepharose XL resin (GE Healthcare) at room temperature. Columns wereequilibrated with buffer A (20 mM Tris-HCl, pH=8.2) at a flow rate of 12mL/min and washed three times with the same buffer. A linear gradientelution was generated by changing from 100% buffer A to 75% buffer B(20mM Tris-Cl, plus 0.2 M NaCl pH8.20) followed by a step gradient of100% buffer B. Upon elution, a fraction collector was used to collectprotein fractions. Protein thus obtained was analyzed by SDS page andstored frozen at −80 ° C.

Human Haemoglobin (SEQ ID NO: 18 or 22, see FIG. 1) can be readilycovalently linked to appropriate drug conjugates, host hydrophobic drugmolecules within the heme binding pocket or even transport smallcytotoxic molecules linked to the heme iron. Hb can be easily modifiedby selective attachment of the appropriate drug conjugate to thecysteine residue in position 93 of the beta chains, the only titratablecystein on the protein surface. The apo-protein solution must be kept atice bath for the duration of the reconstitution process. A 1.5-foldmolar excess of CuT-CPP (Cu-TCPP4,4′,4″,4′″-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)—Cu⁶⁷) in0.1 M NaOH must be added dropwise to the apo-globin solution in 0.2 MKPi buffer at pH 7.0 vortexed quickly at room temperature and thenplaced back in ice bath for 30 minutes. The protein solution must thenbe filtered in a syringe filter before use.

Example 7—Preparation of Transferrin-Compound Complex

The serum was obtained from healthy donor and excess iron was added inthe presence of citrate ions as a chelator and bicarbonate, which isfacilitates for iron binding to transferrin. The reaction mixturecontained 6.5 mg sodium bicarbonate and 153.16 ferric citrate in pH=8,4° C., 1 h per 100 mL of serum. Albumin was subsequently precipitated byRivanol (4%) by adding the alcohol solution to the serum sample in a 3.5V/V ratio at 4° C., and pH=9.4 for 2 h. Then, the solution wascentrifuged at 3000 rpm for 20 min and finally filtered by filter on a0.8 mm syringe filter. Excess Rivanol was subsequently removed bygel-filtration on a Sephadex G-25 column in ammonium sulfate 0.025 M. Afirst precipitation of by saturated ammonium sulfate 50% at pH=6.5 wassubsequently carried out followed by centrifugation at 3000 rpm for 10min (immunoglobulin removal). A second precipitation at 80% saturatedammonium sulfate was then carried out thus allowing recovery oftransferrin the precipitate. Solid precipitate was then dissolved inbuffer of 0.06 M Tris HCl buffer, pH=8, containing 1 M NaCl. Thesolution was dialyzed in the same buffer to allow full removal ofammonium sulfate. Protein solution was then concentrated with acentricon PM50 centrifugal concentrator up to 10-15 mg/ml (as estimatedby Bradford method) and loaded on a Sephadex G-100 gel-filtration column(2.4×80 cm) equilibrated in 1M NaCl, flow rate of 15 ml/h. Transferrinthus obtained was estimated to be 88-90% pure by SDS page. Ion-exchangechromatography by anion exchanger DEAE Sephadex A-50 was then used as afinal polishing step. The transferrin sample was loaded in the columnequilibrated with 0.06 M Tris HCl at pH=8 and eluted by a linearconcentration gradient with elution buffer, 0.3 M Tris HCl, pH=8.Protein purity was higher than 98% with a yield of about 150 mg per 100mL of serum.

Human Holo-transferrin, (SEQ ID NO: 28, FIG. 1) similarly to haemoglobincan be readily covalently linked to appropriate label conjugates. Theapo-protein solution must be kept at ice bath for the duration of thereconstitution process. A 1.5-fold molar excess of CuT-CPP (Cu-TCPP4,4′,4″,4′″-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)—Cu⁶⁷) in0.1 M NaOH must be added dropwise to the apo-globin solution in 0.2 MKPi buffer at pH 7.0 vortexed quickly at room temperature and thenplaced back in ice bath for 30 minutes. The protein solution must thenbe filtered in a syringe filter before use.

Example 8—Obtaining Ferritin Loaded Cells

Obtained cells are incubated in ferritin solution for a time and at theconcentration sufficient to ensure proper ratio of ferritin/cell fortheir full load and also to ensure proper contrast content to obtainproper imaging). The time and concentration may vary depending on thenumber of molecules encapsulated/adsorbed into the ferritin cage, statusof cell activation, condition and number of their intendedadministration.

For example, to ensure proper load with ferritins, cells are incubatedfor 1-4 hrs in ferritin solution 0.2 mg/ml in standard cultureconditions. The frame of ferritin concentration may vary at leastbetween 0.01 and 4 mg/ml as well as incubation time (5 min-6 hrs ormore). Adjusting time and concentration of ferritin load to cells, theinfluence of ferritin and treatment conditions on cell viability shouldbe minded. Cells obtained as stated above very easily uptake ferritinsin a relatively short time (in minutes; FIG. 5). Once they absorbferritins, they do not release it to the culture medium (FIG. 6).

Nevertheless, the person skilled in the art is able to re-adjust theabove conditions and optimize the protocol for the own purposes in theown laboratory.

Example 9—Obtaining Haemoglobin Loaded Cells

Obtained cells are incubated in haemoglobin solution for a time and atthe concentration sufficient to ensure proper ratio of haemoglobin/cellfor their full load and also to ensure proper contrast content to obtainproper imaging. The time and concentration may vary depending on thenumber of molecules linked with the haemoglobin molecule, status of cellactivation, condition and number of their intended administration.

For example, to ensure proper load with haemoglobins, cells areincubated for 1-4 hrs in haemoglobin solution 0.1 mg/ml in standardculture conditions. The frame of haemoglobin concentration may vary atleast between 0.01 and 0.2 mg/ml as well as incubation time (5 min-4 hrsor more). Adjusting time and concentration of haemoglobin load to cells,the influence of ferritin and treatment conditions on cell viabilityshould be minded. Cells obtained as stated above very easily uptakehaemoglobins in a relatively short time (in minutes; FIG. 5).

Nevertheless, the person skilled in the art is able to re-adjust theabove conditions and optimize the protocol for the own purposes in theown laboratory.

Example 10—Obtaining Transferrin Loaded Cells

Obtained cells are incubated in transferrin solution for a time and atthe concentration sufficient to ensure proper ratio of transferrin/cellfor their full load and also to ensure proper contrast content to obtainproper imaging. The time and concentration may vary depending on thenumber of molecules linked with the transferrin molecule, status of cellactivation, condition and number of their intended administration.

For example, to ensure proper load with transferrins, cells areincubated for 1-4 hrs in transferrin solution 0.1 mg/ml in standardculture conditions. The frame of transferrin concentration may vary atleast between 0.01 and 0.2 mg/ml as well as incubation time (5 min-4 hrsor more). Adjusting time and concentration of transferrin load to cells,the influence of transferrin and treatment conditions on cell viabilityshould be minded. Cells obtained as stated above very easily uptaketransferrin in a relatively short time (in minutes; FIG. 5).

Nevertheless, the person skilled in the art is able to re-adjust theabove conditions and optimize the protocol for the own purposes in theown laboratory.

Example 11—Cell Labeling with Radioisotope

In this invention cells were labeled with ¹⁸F-FDG in order to becomeimaged on PET. Cells have been detached from the plate and incubatedwith ¹¹¹F-FDG solution in adequate concentration to ensure the mostoptimal ¹⁸F-FDG uptake by cells allowing their radio-detection at thesite of their accumulation. In this practical invention cells werepre-treated with various cytokine cocktails, growth factors and otherfactors influencing their activation state to enhance their uptake oflabel and incubated in the warm ¹⁸F-FDG solution containing 20-60 MBq.After labeling, cells should be centrifuged and supernatant should beremoved. This step should be repeated until no radioactivity is detectedin the supernatant.

Example 12—Ferritin/Haemoglobin/Transferrin/Label-Leukocyte Complex asUseful Delivery Tool to Tumor

The macrophages from Example 1 prepared as described in Examples 8, 9,10 and 11; monocytes from Example 2 prepared as described in Examples 8,9, 10 and 11, granulocytes from Example 3 prepared as described inExamples 8, 9, 10 and 11, and lymphocytes from Example 4 prepared asdescribed in Examples 8, 9, 10 and 11, very easily transport ferritins,haemoglobins, transferrins and labels to the tumor in the enough amountto be detected using imaging systems (FIGS. 2, 3, 4, 7, and 8).

Example 13—Leukocyte-Protein Carrier Complex as Useful Targeted DrugDelivery Agent to Hypoxic Regions

Macrophages prepared as above are injected into the tail vein of animalwith the tumour (appropriate number of macrophages should be adjusted tothe tumour size, stage of development and presence of metastases). As itis shown on FIGS. 2, 3, 4, 7, 8 and 10 they specifically reach thetumour (after a few hrs) and also disperse in other organs of the wholeanimal (. Moreover, as it is shown on FIG. 9, in hypoxic model they arealso able to migrate to the avascular and hypoxic sites.

For the imaging purposes, 1-50 millions of macrophages were injectedinto the tail vein of mammary or colon cancer tumour-bearing animal.Before, macrophages were pre-labeled with Cell Tracker and loaded withferritin-FITC (as shown in Example 8). Using two-photon imaging of thetumour mass 8 hrs after administration of macrophages the presence ofmacrophages carrying Ferritin-FITC was detected (FIG. 7). Their specifictargeting of tumour but also their migration to other organs was shownusing whole animal body imaging (IVIS) after macrophage pre-labelingusing XenoLight DiR lipophilic DIR cytoplasmic dye (FIG. 8).

Example 14—Leukocyte-Protein Carrier Complex or Labeled Leukocyte asUseful Imaging Tool

The targeted delivery system described in present invention constitutesvery useful imaging tool. As it is presented on FIGS. 4 and 10, afterinjection of 1-50 ml of macrophages loaded with ferritin coupled asdescribed in Example 8 with a contrast agent (in this example:ferrihydrite, however the same results are obtained with isotope, e.g.¹²³I) or labeled with isotope (in this case ¹⁸F-FDG) (FIG. 10) they canbe easily detected by MRI, PET or SPECT. In this example (FIG. 4),mammary-tumour bearing mice were imaged using MRI at 3, 22 and 24 hoursafter i.v. injection of macrophages loaded with ferritin Fh. As it isshown on FIG. 4 the ferritin/macrophage complex is very useful imagingtool. The mouse was treated (at time point 0 h) with macrophages. Thenincreased diameter of blood vessels (arrow) filled with injectedmacrophages (giving significant T2-signal reduction) has been observedand afterword macrophages spread to the tissue (spot-like pattern;arrows). These changes (in the same time points) were observed in allexamined mice.

Macrophages were also labeled with ¹⁸F-FDG (1-50 min) and imaged usingPET at 1 h after i.v. administration to the tumour-bearing mice. Thesemice were inoculated with 4T1 metastatic cell line 3 weeks before theexperiment and metastases in the lungs, liver and spleen werehistopatologically confirmed. At FIG. 10 it is seen that macrophagesmigrated to the regions with metastatic tumours allowing theirvisualization at PET.

While the foregoing written description of the invention enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific embodiment, method, and examples herein. The inventionshould therefore not be limited by the above described embodiment,method, and examples, but by all embodiments and methods within thescope and spirit of the invention.

The present invention also relates to the following aspects andpreferred embodiments of these aspects. The definitions provided abovesimilarly apply to below aspects and embodiments.

-   -   1. Targeted delivery system comprising an activated macrophage        loaded with ferritin carrying a contrast agent.    -   2. The targeted delivery system according to embodiment 1,        wherein the contrast agent is a ferrihydride or an isotope.    -   3. Method of preparation of the targeted delivery system        comprising an activated macrophage loaded with ferritin carrying        a contrast agent comprising steps of    -   a) ferritin purification;    -   b) obtaining ferritin carrying a contrast agent by linking of        ferritin with said contrast agent;    -   c) activation of isolated macrophages;    -   d) incubation of macrophages in solution of ferritin carrying a        contrast agent as obtained in step b) for a time and at the        ferritin concentration sufficient to ensure full load of        ferritin carrying a contrast agent into macrophages.    -   4. The method of embodiment 3, wherein activated macrophages are        bone marrow originated macrophages.    -   5. The method of embodiment 3, wherein activated macrophages are        blood originated macrophages.    -   6. The method of embodiment 3, wherein activated macrophages are        derived from macrophage cell lines.    -   7. The method of any one of embodiments 3-6, wherein activated        macrophages are macrophages polarized towards M1 or M2.    -   8. The method of embodiment 7 wherein activated macrophages have        been polarized towards M2.    -   9. The method of embodiment 7 wherein activated macrophages have        been manipulated with respect to iron metabolism.    -   10. The method of any one of embodiments 3-9, wherein the        contrast agent is a ferrihydride or an isotope.    -   11. Targeted delivery system as defined in any of embodiments 1        or 2 for use as imaging tool.

In a preferred embodiment the present invention does not comprise thesubject-matter of items 1 to 11 above.

1-24. (canceled)
 25. A method for diagnosing a tumor, comprisingadministering to a subject in need thereof, a CD45+ leukocyte cellcomprising within said cell a complex of one or more iron bindingproteins and a label to diagnose the tumor.
 26. The method of claim 25,wherein the label is encapsulated by the one or more iron bindingprotein or multimers thereof.
 27. The method of claim 26, wherein thereis no covalent or non-covalent bond between the one or more iron bindingprotein and the label.
 28. The method of claim 27, wherein the one ormore iron binding protein is ferritin.
 29. The method of claim 25,wherein the CD45+ leukocyte cell is producible from a CD34+hematopoietic precursor cell.
 30. The method of claim 25, wherein theCD45+ leukocyte is selected from the group consisting of a monocyte, adifferentiated monocyte, a lymphocyte and a granulocyte.
 31. The methodof claim 25, wherein (i) the leukocyte is a monocyte, wherein themonocyte is a CD11b⁺ monocyte, (ii) the leukocyte is a differentiatedmonocyte, wherein the differentiated monocyte is selected from the groupconsisting of a macrophage, an activated macrophage, or a dendriticcell, (iii) the leukocyte is a lymphocyte, wherein the lymphocyte isselected from the group consisting of a CD3⁺ and CD4⁺ or CD8⁺ Tlymphocyte, or a CD19⁺, CD20⁺, CD21⁺, CD19⁺ CD20⁺, CD19⁺ CD21⁺, CD20⁺CD21⁺, or CD19⁺ CD20⁺ CD21⁺ B lymphocyte, (iv) the leukocyte is agranulocyte, wherein the granulocyte is selected from the groupconsisting of a neutrophil, an eosinophil and a basophil.
 32. The methodof claim 25, wherein the CD45+ leukocyte is a differentiated monocyte,wherein the differentiated monocyte is an activated macrophage, whereinthe activated macrophage: (i) is producible by in vitro incubation of amonocyte or macrophage with a factor capable of altering expressionmarkers on macrophages; (ii) is characterized by expression of at leastone of following antigens: CD64, CD86, CD16, CD32, high expression ofMHCII, and/or production of iNOS and/or IL-12; (iii) is producible by invitro incubation of a monocyte or macrophage with a factor capable ofinducing the ability of the macrophage to phagocytose; (iv) ischaracterized by expression of at least one of following antigens:CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC-motivechemokine receptor 4 (CXCR4), CD163, and/or T cell immunoglobulin-domainand mucin-domain 2 (TIM-2), and/or show low expression of MHCII; (v) hasthe ability to phagocytose; and/or (vi) is capable of cytokinesecretion, or production of inducible nitric oxide synthetase (iNOS) (orother pro-inflammatory compounds), arginase or otherimmunosuppressive/anti-inflammatory compounds.
 33. The method of claim25, wherein (i) the M1 inducer is selected from the group consisting ofLPS, INF-γ, GM-CSF and viral and bacterial infection; or (ii) the M2inducer is selected from the group consisting of IL-4, IL-10, IL-13,immune complex of an antigen and antibody, IgG, heat activatedgamma-globulin, glucocorticosteroid, TGF-β, IL-1R, CCL-2, IL-6, M-CSF,PPARγ agonist, Leukocyte inhibitory factor, adenosine, helminth andfungal infection.
 34. The method of claim 25, wherein the CD45+leukocyte is a monocyte, wherein the monocyte: (i) is producible from aCD34⁺ hematopoietic precursor cell; (ii) is producible by in vitroincubation of monocytes with at least one inducer; (iii) ischaracterized by expression of at least one of the following antigens:TfR⁺, CD163⁺, TIM-2⁺, CD14⁺, CD16⁺, CD33⁺, and/or CD115⁺; (iv) ischaracterized by expression of at least one of the following antigens:TfR⁺, CD163⁺, TIM-2⁺, CXCR4⁺, CD14⁺, and/or CD16⁺; and/or (v) has theability to phagocytose.
 35. The method of claim 25, wherein: (i) the M1inducer is selected from the group consisting of LPS, INF-γ, GM-CSF orviral or bacterial infection; (ii) the M2 inducer is selected from thegroup consisting of IL-4, IL-10, IL-13, immune complex of an antigen andantibody, IgG, heat activated gamma-globulins, Glucocorticosteroids,TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonist, Leukocyte inhibitoryfactor, cancer-conditioned medium, cancer cells, adenosine and helminthor fungal infection.
 36. The method of claim 25, wherein the CD45+leukocyte is a lymphocyte, wherein the lymphocyte: (i) is obtainablefrom blood, spleen, or bone marrow or is producible from a CD34⁺precursor cell; (ii) is an immunologically competent lymphocyte; (iii)expresses antigen specific T cell receptors; and/or (iv) ischaracterized by expression of at least one of the following antigens:(a) CD3⁺ and CD4⁺ or CD8⁺ or (b): CD19⁺, CD20⁺, CD21⁺, CD19⁺ CD20⁺,CD19⁺ CD21⁺, CD20⁺ CD21⁺, or CD19⁺ CD20⁺ CD21⁺ antigen.
 37. The methodof claim 25, wherein the CD45+ leukocyte is a granulocyte, wherein thegranulocyte: (i) is obtainable from blood, spleen or bone marrow orproducible from a CD34⁺ precursor cell; (ii) is characterized byexpression of at least one of the following CD66b⁺ and/or CD193⁺; (iii)is a polymorphonuclear leukocyte characterized by the presence ofgranules in their cytoplasm; and/or (iii) is characterized by expressionof at least one of the following: TfR⁺, CD163⁺, TIM-2⁺, and/or CXCR4⁺.38. The method of claim 25, wherein the one or more iron binding proteinis selected from the group consisting of ferritin, haemoglobin,haemoglobin-haptoglobin complex, hemopexin, transferrin, andlactoferrin.
 39. The method of claim 25, wherein the label is selectedfrom the group consisting of a fluorescent dye, a fluorescence emittingisotope, a radioisotope, a detectable polypeptide, a nucleic acidencoding a detectable polypeptide, and a contrast agent.
 40. The methodof claim 25, wherein the label comprises a chelating agent which forms acomplex with divalent or trivalent metal cations.
 41. The method ofclaim 40, wherein the chelating agent is selected from the groupconsisting of 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid(DOTA), ethylenediaminetetraacetic acid (EDTA),1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),triethylenetetramine (TETA), iminodiacetic acid,Diethylenetriamine-N,N,N′,N′,N′″-pentaacetic acid (DTPA) and6-Hydrazinopyridine-3-carboxylic acid (HYNIC).
 42. The method of claim39, wherein the label is a contrast agent that comprises a paramagneticagent or ferrihydride.
 43. The method of claim 39, wherein the label isa radioisotope or fluorescence emitting isotope selected from the groupconsisting of alpha radiation emitting isotopes, gamma radiationemitting isotopes, Auger electron emitting isotopes, X-ray emittingisotopes, fluorescent isotopes, fluorescence emitting isotopes, as wellas conjugates and combinations of above with proteins, peptides, smallmolecular inhibitors, antibodies or other compounds.
 44. The method ofclaim 39, wherein the label is a fluorescence dye selected from thegroup consisting of the following classes of fluorescent dyes: Xanthens,Acridines, Oxazines, Cynines, Styryl dyes, Coumarines, Porphines,Metal-Ligand-Complexes, Fluorescent proteins, Nanocrystals, Perylenesand Phtalocyanines as well as conjugates and combinations of theseclasses of dyes.
 45. The method of claim 39, wherein the label is adetectable polypeptide, wherein the detectable polypeptide is anautofluorescent protein or any structural variant thereof with analtered adsorption and/or emission spectrum.
 46. The method of claim 25,wherein: (i) bond(s) between the iron binding protein(s) and the labelcomprised in the complex are covalent and/or non-covalent; and/or (ii)the label comprised in the complex is entrapped/encapsulated by the oneor more iron binding proteins or multimers thereof.
 47. A diagnosticcomposition comprising an isolated targeted delivery system comprising aCD45+ leukocyte cell comprising within said cell a complex of one ormore iron binding proteins and a label and a pharmaceutically acceptablecarrier and/or suitable excipient(s).
 48. The diagnostic composition ofclaim 47, wherein the label is encapsulated by the iron binding proteinor multimers thereof.
 49. The diagnostic composition of claim 48,wherein there is no covalent or non-covalent bond between the ironbinding protein and the label.
 50. The diagnostic composition of claim49, wherein iron binding protein is ferritin.